U.S. patent application number 12/842468 was filed with the patent office on 2010-12-09 for regulatory nucleic acid elements.
This patent application is currently assigned to Boehringer Ingelheim Pharma GmbH & Co.KG. Invention is credited to Barbara ENENKEL, Kerstin SAUTTER.
Application Number | 20100311121 12/842468 |
Document ID | / |
Family ID | 37441548 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100311121 |
Kind Code |
A1 |
ENENKEL; Barbara ; et
al. |
December 9, 2010 |
REGULATORY NUCLEIC ACID ELEMENTS
Abstract
The invention relates to DNA-sequences, especially
transcription- or expression-enhancing elements (TE elements) and
their use on an expression vector in conjunction with an enhancer,
a promoter, a product gene and a selectable marker. The invention
describes Sequence No. 1 and TE elements TE-01, -02, -03, -04, -06,
-07, -08, -10, -11 or -12. Because of their small size, TE-06,
TE-07 or TE-08 are particularly preferred. Sequence No. 1
originates from a sequence region located upstream from the coding
region of the Ub/S27a gene from CHO cells. TE elements bring about
an increase in the expression of the product gene, particularly
when stably integrated in the eukaryotic genome, preferably the
CHO-DG44 genome. Chromosomal positional effects are thereby
overcome, shielded or cancelled out. In this way the proportion of
high producers in a transfection mixture and also the absolute
expression level are increased up to seven-fold.
Inventors: |
ENENKEL; Barbara;
(Warthausen, DE) ; SAUTTER; Kerstin; (Biberach,
DE) |
Correspondence
Address: |
MICHAEL P. MORRIS;BOEHRINGER INGELHEIM USA CORPORATION
900 RIDGEBURY ROAD, P. O. BOX 368
RIDGEFIELD
CT
06877-0368
US
|
Assignee: |
Boehringer Ingelheim Pharma GmbH
& Co.KG
Ingelheim am Rhein
DE
|
Family ID: |
37441548 |
Appl. No.: |
12/842468 |
Filed: |
July 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11762240 |
Jun 13, 2007 |
|
|
|
12842468 |
|
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 435/358; 435/455; 536/23.1 |
Current CPC
Class: |
C12N 2830/85 20130101;
C12N 2830/46 20130101; C12N 2800/107 20130101; A61P 43/00 20180101;
C07K 14/47 20130101; C12N 15/63 20130101; C12N 15/85 20130101 |
Class at
Publication: |
435/69.1 ;
536/23.1; 435/320.1; 435/325; 435/358; 435/455 |
International
Class: |
C12N 15/85 20060101
C12N015/85; C07H 21/04 20060101 C07H021/04; C12N 5/10 20060101
C12N005/10; C12N 15/09 20060101 C12N015/09; C12P 21/00 20060101
C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 26, 2006 |
EP |
06117862.0 |
Claims
1. A nucleic acid comprising TE-13 (SEQ ID No. 15) or a fragment of
TE-13 (SEQ ID No. 15) or the complementary nucleotide sequences
thereof or a derivative of TE-13 (SEQ ID No. 15) or a fragment
thereof or the complementary nucleotide sequences thereof, wherein
on chromosomal integration said nucleic acid leads to an increase
in the transcription or expression of a gene of interest in an
expression system.
2. The nucleic acid according to claim 1, wherein said nucleic acid
comprises TE-08 (SEQ ID No. 10) or a fragment of TE-08 (SEQ ID No.
10) or the complementary nucleotide sequences thereof or a
derivative of TE-08 (SEQ ID No. 10) or a fragment thereof or the
complementary nucleotide sequences thereof, wherein on chromosomal
integration said nucleic acid leads to an increase in the
transcription or expression of a gene of interest in an expression
system.
3. The nucleic acid according to claim 2, wherein said nucleic acid
comprises SEQ ID No. 1 or a fragment of SEQ ID No. 1 or the
complementary nucleotide sequences thereof or a derivative of SEQ
ID No. 1 or a fragment thereof or the complementary nucleotide
sequences thereof, wherein on chromosomal integration said nucleic
acid leads to an increase in the transcription or expression of a
gene of interest in an expression system, wherein said fragment
comprises at least one sequence region from the nucleic acid region
between 1 bp and 1578 bp (in relation to SEQ ID No. 1).
4. The nucleic acid according to claim 1, wherein the increase in
the transcription or expression of a gene of interest in an
expression system in comparison to a control which does not contain
a TE element, is determined by measuring the product titre by
ELISA.
5. The nucleic acid according to claim 1, wherein said nucleic acid
hybridises under stringent conditions (a) with the region of
nucleic acid sequence TE-13 (SEQ ID No. 15); or (b) the
complementary nucleic acid sequences thereof; or (c) a nucleic acid
sequence which has at least about 70% sequence identity with a
sequence of (a) or (b).
6. The nucleic acid according to claim 5, wherein said nucleic acid
has a length of at least 1015 by (=length TE-8, SEQ ID No. 10).
7. The nucleic acid according to claim 5, wherein said nucleic acid
has a length of at least 511 by (=length TE-13, SEQ ID No. 15).
8. The nucleic acid according to claim 2, wherein said nucleic acid
hybridises under stringent conditions (a) with the region of
nucleic acid sequence TE-08 (SEQ ID No. 10); or (b) the
complementary nucleic acid sequences thereof; or (c) a nucleic acid
sequence which has at least about 70% sequence identity with a
sequence of (a) or (b).
9. The nucleic acid according to claim 1, wherein said nucleic acid
is a fragment or derivative of TE-01 (SEQ ID No. 3).
10. The nucleic acid according to claim 9, wherein said nucleic
acid is TE-13 (SEQ ID No. 15), TE-14 (SEQ ID No. 16), TE-15 (SEQ ID
No. 17), TE-16 (SEQ ID No. 18), TE-17 (SEQ ID No. 19) or TE-18 (SEQ
ID No. 20).
11. The nucleic acid according to claim 1, wherein said nucleic
acid or a fragment or derivative thereof is an isolated nucleic
acid.
12. The nucleic acid according to claim 1, wherein said nucleic
acid is linked to a heterologous sequence.
13. An isolated nucleic acid selected from the group consisting of
TE-00 (SEQ ID No. 2), TE-01 (SEQ ID No. 3), TE-02 (SEQ ID No. 4),
TE-03 (SEQ ID No. 5), TE-04 (SEQ ID No. 6), TE-06 (SEQ ID No. 8),
TE-07 (SEQ ID No. 9), TE-08 (SEQ ID No. 10), TE-10 (SEQ ID No. 12),
TE-11 (SEQ ID No. 13), TE-12 (SEQ ID No. 14), TE-13 (SEQ ID No.
15), TE-14 (SEQ ID No. 16), TE-15 (SEQ ID No. 17), TE-16 (SEQ ID
No. 18), TE-17 (SEQ ID No. 19), TE-18 (SEQ ID No. 20) and TE-21
(SEQ ID No. 21).
14. The isolated nucleic acid according to claim 13, wherein said
nucleic acid is TE-06 (SEQ ID No. 8).
15. The isolated nucleic acid according to claim 13, wherein said
nucleic acid is TE-08 (SEQ ID No. 10).
16. The nucleic acid according to claim 13, wherein said nucleic
acid is TE-13 (SEQ ID No. 15).
17. A eukaryotic expression vector comprising a nucleic acid
according to claim 1.
18. The eukaryotic expression vector according to claim 17, wherein
said expression vector further comprises a promoter and a
heterologous gene of interest.
19. The eukaryotic expression vector according to claim 18, wherein
said expression vector further comprises an enhancer.
20. The eukaryotic expression vector according to claim 18, wherein
said expression vector further comprises a selectable marker.
21. The eukaryotic expression vector according to claim 20, wherein
said selectable marker is DHFR, Neo, or Neo F240I.
22. The eukaryotic expression vector according to claim 17, wherein
said expression vector comprises a combination of several identical
or different said nucleic acids, wherein one or more said nucleic
acids are positioned in front of (i.e. 5' of) said gene of
interest, or one or more said nucleic acids are positioned behind
(i.e. 3' of) said gene of interest, or one or more said nucleic
acids are positioned in front of and behind said gene of
interest.
23. The eukaryotic expression vector according to claim 22, wherein
said nucleic acids are TE-06 (SEQ ID No. 8), TE-21 (SEQ ID No. 21)
or TE-08 (SEQ ID No. 10).
24. The eukaryotic expression vector according to claim 22, wherein
said combination comprises a TE-08 nucleic acid (SEQ ID No. 10)
followed by a TE-06-nucleic acid (SEQ ID No. 8).
25. The eukaryotic expression vector according to claim 22, said
combination comprises one or more TE-08-nucleic acid(s) (SEQ ID No.
10) positioned in front of (i.e. 5' of) and one or more
TE-08-nucleic acid(s) (SEQ ID No. 10) positioned behind (i.e. 3'
of) said gene of interest.
26. The eukaryotic expression vector according to one of claim 17,
wherein said expression vector comprises one or more said nucleic
acids distributed over 2 plasmids.
27. Method of producing a eukaryotic expression vector comprising
the step of integrating a nucleic acid according to claim 1 in an
expression vector.
28. A eukaryotic host cell comprising a eukaryotic expression
vector according to claim 17.
29. The eukaryotic host cell according to claim 28, wherein said
cell is a high producer, wherein said cell has a higher specific
productivity than a comparable eukaryotic host cell lacking a TE
element, wherein said host cell has an expression level which is
increased up to two-fold, three-fold, four-fold, five-fold,
six-fold, seven-fold or ten-fold or one which is increased more
than two-fold, more than three-fold, more than four-fold, more than
five-fold, more than seven-fold or more than ten-fold when compared
to said cell lacking a TE element.
30. The eukaryotic host cell according to claim 28, wherein said
expression vector is stably integrated in the genome of said
cell.
31. The eukaryotic host cell according to claim 28 wherein said
host cell is a mammalian cell.
32. The eukaryotic host cell according to claim 31, wherein said
host cell is a CHO, NS0, Sp2/0-Ag14, BHK21, BHK TK.sup.-, HaK,
2254-62.2 (BHK-21-derivative), CHO-K1, CHO-DUKX(=CHO duk.sup.-,
CHO/dhfr.sup.-), CHO-DUKX B1, CHO-DG44, CHO Pro-5, V79, B14AF28-G3,
or CHL cell.
33. The eukaryotic host cell according to claim 32, wherein said
host cell is a CHO-DG44 cell.
34. The eukaryotic host cell according to claim 28, wherein said
cell further comprises an anti-apoptosis gene.
35. The eukaryotic host cell according to claim 34, wherein said
anti-apoptosis gene is BCL-xL, BCL-2, BCL-w, BFL-1, A1, MCL-1, BOO,
BRAG-1, NR-13, CDN-1, CDN-2, CDN-3, BHRF-1, LMW5-HL or CED-9.
36. Method of developing a high-producing stably transfected
eukaryotic host cell line comprising the steps of: (a) integrating
at least one nucleic acid according to claim 1 in a eukaryotic
expression vector containing a gene of interest, (b) transfecting a
eukaryotic host cell with an expression vector obtained in step
(a), (c) selecting a highly-productive transfected host cell.
37. The method according to claim 36, further comprising an
amplification step.
38. The method according to claim 37, further comprising a cloning
step.
39. Method of preparing and selecting recombinant mammalian cells
comprising the steps of: (a) transfecting the host cells with a
gene that codes for a protein/product of interest, a
neomycin-phosphotransferase, and the amplifiable selectable marker
DHFR, wherein in order to enhance the transcription or expression
said gene of interest is functionally linked to at least one
nucleic acid according to claim 1, (b) cultivating the cells under
conditions which enable expression of said genes, (c) selecting
these co-integrated genes by cultivating the cells in the presence
of a selecting agent in a hypoxanthine/thymidine-free medium, and
(d) amplifying these co-integrated genes by cultivating the cells
in the presence of a selecting agent which allows the amplification
of at least the amplifiable selectable marker gene.
40. The method according to claim 39, wherein said transfected
cells are cultivated in hypoxanthine/thymidine-free medium,
supplemented with at least 200 .mu.g/mL G418, in the absence of
serum and with the addition of increasing concentrations of
methotrexate (MTX).
41. The method according to claim 40, wherein the concentration of
MTX in the first amplification step is at least 100 nM or at least
250 nM and is increased stepwise to 1 .mu.M or above.
42. The method according to claim 39, further comprising improving
the glycosylation of said protein of interest.
43. The method according to claim 39, wherein said host cell is a
mammalian cell.
44. The method according to claim 43, wherein said mammalian cell.
is a CHO, NS0, Sp2/0-Ag14, BHK21, BHK TK.sup.-, HaK, 2254-62.2
(BHK-21-derivative), CHO-K1, CHO-DUKX(=CHO duk.sup.-,
CHO/dhfr.sup.-), CHO-DUKX B1, CHO-DG44, CHO Pro-5, V79, B14AF28-G3,
CHL cell.
45. The method according to claim 44, wherein said mammalian cell
is a CHO-DG44 cell.
46. The method according to claim 39, wherein the expression vector
comprises the selectable marker DHFR.
47. The method according to claim 39, wherein the proportion of
high producers is increased up to two-fold, three-fold, four-fold,
five-fold, six-fold, seven-fold or ten-fold or more than two-fold,
more than three-fold, more than four-fold, more than five-fold,
more than seven-fold or more than ten-fold.
48. Method of preparing a biopharmaceutical product comprising the
steps of: (a) integrating at least one nucleic acid according to
claim 1 in a eukaryotic expression vector containing a gene of
interest, (b) transfecting a eukaryotic host cell with an
expression vector obtained in step (a), (c) selecting a
highly-productive transfected host cell obtained in step (b) and
(d) cultivating the highly-productive transfected host cell
selected in step (c) under conditions which allow expression of the
gene(s) of interest.
49. The method according to claim 48, further comprising the step
of: (e) harvesting and purifying said protein of interest.
50. Kit consisting of a nucleic acid according to claim 1, an
expression vector and a host cell.
51. The kit according to claim 50, further comprising a
transfection reagent.
Description
[0001] This application claims priority benefit from European
application EP 06 117 862.0, filed Jul. 26, 2006, which is
incorporated herein in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to cis-active nucleic acid sequences,
so-called TE elements. The TE elements preferably originate from
the CHO genome. Their use in expression vectors, for example, in
stable cell populations permits at least twice as high an
expression of a gene of interest in a desired chromosome locus
compared with vectors previously used.
BACKGROUND TO THE INVENTION
[0003] Mammalian cells are the preferred host cells for the
production of complex biopharmaceutical proteins as the
post-translational modifications are human-compatible both
functionally and from a pharmacokinetic point of view. The main
relevant cells types are hydridomas, myelomas, CHO (Chinese Hamster
Ovary) cells and BHK (Baby Hampster Kidney) cells. The host cells
are increasingly cultivated under serum- and protein-free
production conditions. The reasons for this are the associated
reduction in costs, the reduced interference in the purification of
the recombinant protein and the reduction of the potential for
introducing pathogens (e.g. prions and viruses). The use of CHO
cells as host cells is becoming more and more widespread as these
cells adapt to suspension growth in serum- and protein-free medium
and moreover are regarded and accepted as safe production cells by
the regulatory bodies.
[0004] In order to produce a stable mammalian cell line which
expresses a heterologous gene of interest, the heterologous gene is
generally introduced into the desired cell line together with a
selectable marker gene, such as neomycin phosphotransferase (NPT),
by transfection. The heterologous gene and the selectable marker
gene can be expressed in a host cell, starting from an individual
or separate co-transfected vectors. Two to three days after the
transfection the transfected cells are transferred into medium
containing a selective agent, e.g. G418 when using the
neomycin-phosphotransferase gene (NPT gene) and cultivated for a
few weeks under these selective conditions. The emergent resistant
cells which have integrated the exogenous DNA can be isolated and
investigated for the expression of the desired gene product (gene
of interest).
[0005] For biopharmaceutical production, cell lines with a high
stable productivity are required. The expression vectors for
production cells are equipped with strong, usually constitutively
expressing promoters and enhancers such as CMV enhancer and
promoter, for example, to allow high product expression. As the
expression of the product has to be guaranteed over the longest
possible time, cells are selected which have the product gene
stably integrated in their genome. This is done with selectable
markers such as e.g. neomycin-phosphotransferase (NPT) and
dihydrofolate reductase (DHFR).
[0006] By the chance integration of the expression vectors in the
host cell genome, cells are obtained with different levels of
expression of the desired gene product, as its expression is not
determined solely by the strength of the previous promoter or the
promoter/enhancer combination. The chromatin structure present at
the integration site can affect the level of expression both
negatively and positively. Increasingly, therefore, cis-active
elements which positively influence the expression at the chromatin
level are integrated in expression vectors. These include locus
control regions (LCR) which occur for example in the 5' region of
the .beta.-globin genes (Li et al., 2002) and in the 3' region of
the TCR.alpha. gene. They cause high tissue-specific expression of
a coupled transgene in the chromatin, which is characterised by its
independence of position and dependence on copy number. These
properties indicate that LCRs are capable of opening chromatin in
their native tissue (Ortiz et al., 1997). There are various forms
of .beta.-thalassaemia in which the .beta.-globin locus is intact
but is not expressed. The reason for the lack of expression is a
major deletion in the 5' direction of the .beta.-globin genes. The
deletion of this .beta.-globin LCR leads to a closed chromatin
configuration which extends over the entire locus and leads to
suppression of gene expression (Li et al., 2002). LCRs colocalise
with DNAse I-hypersensitive sites (HS) in the chromatin of
expressing cells. The occurrence of HS also indicates open
chomatin. The HS contain a series of different general and
tissue-specific binding sites for transcription factors. By the
interaction of the transcription factors with the DNA the open
chromatin structure of HS is produced (Li et al., 2002). Many LCRs
are known to be made up of a number of HS the functions of which
can be more or less separated from one another. The TCR.alpha. gene
for example is expressed under endogenous control only in T-cell
tissue. The locus exists in various chromatin modes depending on
the tissue and expression status. In the 3' region it has a locus
control region which has 8 HS. HS 2-6, a 6 kb partial fragment of
the LCR, has a chromatin-opening activity and is not tissue
specific. The tissue specificity is imparted to the T-cell-specific
expression in the thymus by HS7, 8 and 1 (3 kb). Only in the
complete combination of all the HS is the TCR.alpha.LCR
functionally complete (Ortiz et al., 1997). A more precise
subdivision and specification of the individual HS functions of the
TCR.alpha.LCR can be found in (Ortiz et al., 1999). This Example
shows that LCRs are functionally very complex and may be made up of
different control elements such as enhancers, silencers and
isolators. Other Examples of the division of the LCR functions
between various domains are the TCR.gamma. locus and .beta.-globin
locus. The former is made up of the DNAse I-hypersensitive site HsA
and the enhancer 3'E.sub.c.gamma.l. The TCR.gamma.-LCR, in addition
to having its usual functions, is also thought to play a part in
the recombination of the TCR.gamma. genes (Baker et al., 1999). The
.beta.-globin locus has five HS with distinguishable functions
which also require the tissue-specific promoter in order to
function fully. LCRs could also play another important role in the
tissue-specific demethylation of DNA, as DNA methylation results in
a closed chromatin structure and the inactivation of genes. A
mechanism of activity which activates gene expression by increased
histone acetylation would also be possible (Li et al., 2002).
[0007] Scaffold/Matrix Attachment Regions (S/MARs) are DNA
sequences which bind with a high affinity in vitro to components of
the matrix or the scaffold of the cell nucleus. They form the
structural and possibly also functional boundaries of chromatin
domains (Zahn-Zabal et al., 2001). S/MARs are capable of
interacting with enhancers and of locally increasing the
accessibility of the DNA in the chromatin and in this way can
increase the expression of stably integrated heterologous genes in
cell lines, transgenic animals and plants (Klehr et al., 1991;
Stief et al., 1989; Jenuwein et al., 1997; Zahn-Zabal et al.,
2001). However they cannot totally shield a chromosomal locus from
nearby elements in order to allow position-independent expression
(Poljak et al., 1994). The effect of the MARs can be used to
increase the proportion of (highly) expressing cell clones or
transgenic animals in a transfection experiment (McKnight et al.,
1992; Zahn-Zabal et al., 2001). However, MARs have also been
reported which do not impart high expression but play an important
part in the correct regulation of development-specific genes
(McKnight et al., 1992).
[0008] Isolators are defined as a neutral boundary between
neighbouring regions which influence one another, e.g. between
active and inactive chromatin (boundary elements). They may
restrict the effect of enhancers or isolate entire DNA domains
against them and shield stably transfected reporter genes from
positional effects (Bell and Felsenfeld, 1999; Udvardy, 1999).
Thus, these elements render the expression independent of the
genomic position. They may also prevent the silencing of transgenes
in the absence of selection pressure (Pikaart et al., 1998).
Another presumed function of isolators is the restriction of
replication territories (Bell and Felsenfeld, 1999). The first
isolators that were described are scs and scs' from Drosophila.
They constitute the boundary for the hsp70 heat shock genes and
suppress positional effects (Udvardy et al., 1985).
[0009] As another element with an isolating function, a GC-rich
fragment from the dhfr gene (Chinese Hamster) was found, containing
CpG islands (Poljak et al., 1994). The fragment on its own
exhibited no influence whatever on reporter gene expression.
Situated between an expression promoting SAR and the reporter gene,
however, the fragment was able to substantially prevent the
expression-enhancing effect of the SAR element. Possibly, this
GC-rich fragment blocks the chromatin-opening mechanism of the SAR
element and consequently acts as an isolator. Elements with
extended CPG islands are methylated with a higher probability as
they are recognised by a DNA methyltransferase which converts
cytosine into 5-methylcytosine. Consequently, inactive chomatin is
formed (Poljak et al., 1994).
[0010] Aronow and colleagues defined in the first intron of the
human ADA gene (Adenosin deaminase) a new regulatory element which
substantially contributes to expression which is dependent on gene
copy number but independent of position (Aronow et al., 1995). The
element is up to 1 kb in size and only functional when it flanks a
200 by T-cell-specific enhancer. If only one of the two segments is
present or if the segments are wrongly arranged in their sequence
and orientation, the element is non-functional as this prevents the
formation of DNAse I-hypersensitive sites on the enhancer.
[0011] In their Patent WO 02/081677 the firm Cobra Therapeutics
describe another chromatin-influencing element. The Ubiquitous
Chromatin Opening Elements (UCOEs) are responsible for an open
chromatin structure in chromosomal regions with ubiquitously
expressed household genes (human hnRNP A2 gene, human .beta.-actin
gene, human PDCD2 gene). All these genes have CpG-rich islands in
the untranslated regions which are relatively weakly methylated.
The absence of methylation of CpG islands indicates that there is
active chromatin at this point. The UCOEs help to provide a
strength of expression which is independent of the genomic
environment and the nature of the cell or tissue.
[0012] The firm Immunex also describes cis-active DNA sequences
which bring about an increase in expression (U.S. Pat. No.
6,027,915, U.S. Pat. No. 6,309,851). The element referred to as the
expression augmenting sequence element (EASE) demands a high
expression of recombinant proteins in mammalian cells, is not
active in transient expression systems and does not have the
typical sequence properties found in LCRs and S/MARs. It is also
not a sequence which codes for a trans-activating protein as it
does not contain an open reading frame. The fragment is 14.5 kb
long, originates from the genomic DNA of CHO cells and can increase
the expression of a stably integrated reporter gene eight fold.
Over 50% of the activity of the element is restricted to a 1.8 kb
long segment, while the first 600 base pairs of this segment are
essential for correct function. An additional property of sequence
sections with a high EASE activity is the presence of a number of
HMG-1(Y) binding sites. HMG-I(Y) proteins belong to the family of
the high mobility group non-histone chromatin proteins. They are
also referred to as "architechtonic transcription factors" and form
a new category of trans-regulators of mammalian genes. HMG-I(Y)
proteins recognise 80-rich sequences and bind to their so-called AT
hooks (DNA-binding domains) in the small DNA fork. This can lead to
local changes in the DNA topology and consequently to altered gene
expression.
[0013] The authors of U.S. Pat. No. 6,309,841 presume that the
effects of EASE are connected with the MTX-induced amplification of
the integrated plasmid. In MTX-induced gene amplification,
so-called breakage fusion bridge cycles occur. It is easy to
imagine a role for the HMG-I(Y) proteins in the structural
alteration of the DNA which lead to the formation and removal of
the DNA breakages.
[0014] Other elements for increasing gene expression in mammalian
cells are described in Kwaks et al., 2003. These so-called STAR
elements (Stimulatory and Anti-Repressor Elements) originate from
the screening of a human gene library with 500 to 2100 by DNA
fragments. The screening was carried out using a specially designed
reporter plasmid. The expression of the reporter gene was only
possible when it was functionally linked with an anti-repressor
element from the human gene bank. With the STAR elements thus
obtained the authors were able to protect transgenes from
positional effects in the genome of mammalian cells. A comparison
with the mouse genome showed that the majority of these STAR
elements occur in both the human and the murine genome and are
highly conserved within these two species.
[0015] A major problem in establishing cell lines with a high
expression of the desired protein arises from the random and
undirected integration of the recombinant vector in
transcription-active or -inactive loci of the host cell genome. As
a result, a population of cells is obtained which show completely
different expression rates for the heterologous gene, while the
productivity of the cells generally follows a normal distribution.
In order to identify cell clones which have a very high expression
of the heterologous gene of interest it is therefore necessary to
check and test a number of clones, resulting in high expenditure of
time, labour and costs. Attempts at improving the vector system
used for the transfection are therefore directed at even allowing
or increasing the transcription of a stably integrated transgene by
the use of suitably cis-active elements. The cis-active elements
which act at the chromatin level include for example the locus
control regions, scaffold-matrix attachment regions, isolators,
etc., already described. Some of these elements shield certain
genes from the influences of the surrounding chromatin. Others
exhibit an enhancer-like activity, although this is restricted to
stably integrated constructs. Yet other elements combine several of
these functions in themselves. Often it is not clearly possible to
assign them precisely to a specific group. In stable cell lines the
expression of the transgenic product gene thus underlies
chromosomal positional effects to a considerable extent. This
phenomenon is based on the influence of the chromatin structure
and/or the presence of intrinsic regulatory elements at the
integration site of the foreign DNA. This leads to very variable
expression levels. During the selection of cells, therefore,
frequently clones with a very low or completely absent product
expression are frequently produced. These chromosomal positional
effects are also the reason why the generation of stable production
cell lines which express a high level of a therapeutic protein is
generally a time consuming, high-capacity and expensive process.
Stable cell lines with high productivity are usually produced by
selection with positive selectable markers, frequently combined
with agent-induced gene amplification (e.g. dihydrofolate
reductase/methotrexate or glutamine
synthetase/methioninesulfoximine). The pools and clones which are
produced with this selection strategy are investigated for high and
stable is expression in a complex screening process. The majority
of the clones produce no or only average amounts of product and
only a few are high producers. The proportion of high producers in
a mixed population can be increased, for example, by a mutation in
the selectable marker (Sautter and Enenkel, 2005, WO 2004/050884).
However, it is desirable to further increase the specific
productivity of each individual clone as well as the proportion of
high producers within a transfected cell population.
[0016] The specific productivity of stably transfected cells,
particularly CHO- or other production-relevant cells, and the
proportion of high producers in a transfection batch should be
increased. This should result in the last analysis in a more
efficient cell line development. Consequently more and higher
producing cell lines could be established in a shorter time and
thus save on labour, time and costs.
SUMMARY OF THE INVENTION
[0017] The present invention relates to regulatory nucleic acids,
particularly a nucleic acid having SEQ ID No. 1, known as a "TE
element", or a fragment or derivative thereof, which leads to an
increase in the transcription or expression of a gene of interest
in stably transfected cells. Surprisingly, it has been shown that
the use of a TE element of this kind on an expression vector in
conjunction with a promoter, a product gene, a selectable marker
and optionally an enhancer in stable integration into a host
genome, such as the CHO-DG44 genome, for example, overcomes,
shields or cancels out the chromosomal positional effects. As a
result, both the proportion of high producers in a transfection
batch and also the absolute expression level are increased.
[0018] The invention further relates to expression vectors which
contain transcription- or expression-increasing regions, fragments
or derivatives of SEQ ID No. 1, preferably the TE elements TE-00
(SEQ ID No. 2), TE-01 (SEQ ID No. 3), TE-02 (SEQ ID No. 4), TE-03
(SEQ ID No. 5), TE-04 (SEQ ID No. 6), TE-06 (SEQ ID No. 8), TE-07
(SEQ ID No. 9), TE-08 (SEQ ID No. 10), TE-10 (SEQ ID No. 12), TE-11
(SEQ ID No. 13), TE-12 (SEQ ID No. 14). In view of their small size
TE-06, TE-07 and TE-08 re particularly preferred.
[0019] SEQ ID No. 1 originates from a sequence region located
upstream of the coding region of the ubiquitin/S27a gene, which was
isolated from CHO-cells, the gene coding for an essential protein
in the ribosome metabolism of the cell.
[0020] Compared with the expression vectors used hitherto, the
additional introduction of the cis-active TE elements into
expression vectors results in a productivity of stably transfected
cell pools, particularly CHO-DG44 cell pools, which is up to seven
times higher. In transient transfections of CHO-DG44 cell pools, on
the other hand, no increase in productivity can be achieved by
introducing the TE elements. Thus, the increase in productivity
observed in the stable cell pools is not based on an enhancer
present in the TE elements. Thus, chromosomal integration is
absolutely essential for the increase in productivity caused by TE
elements. This is an indication that TE elements may suppress,
shield or cancel out negative chromosomal positional effects. As a
result, cis-active elements have been produced and identified which
are characterised by their particular suitability for selecting and
enriching high producing cells and are therefore capable of
reducing the expenditure on time, costs and capacity in the
isolation and identification of high producing clones.
[0021] Possible applications for the invention include the
development of high producing cell lines as required for example in
the manufacture of biopharmaceuticals, in analytical cell-based
assays, in high throughput screenings of substances or in the
production of recombinant protein products for NMR spectroscopy,
other assays, etc. Because of the higher specific productivity and
the reduction of cells which express little or no product, more and
higher producing cell lines can be established in a shorter time
and thus labour and costs can be saved. Other possible application
are the production of robust improved host cell lines (e.g. the
introduction of anti-apoptosis or glycosilation genes), transgenic
animals or plants and in gene therapy.
[0022] The invention does not arise from the prior art.
[0023] The nucleic acid with SEQ ID No. 1 is a nucleic acid
sequence isolated from the genome of Chinese hamsters (Cricetulus
griseus). It comes from a sequence region located upstream of the
coding region of the ubiquitin/S27a gene.
[0024] The nucleic acid with SEQ ID No.1 has an average GC content
of 44% and does not contain any lengthy passages of GC repeats.
This GC content is comparable with the average GC content of about
40% described for genomic DNA of mammals (Delgado et al.,
1998).
[0025] Parts of the nucleic acid with SEQ ID No.1 have already been
described in WO 97/15664: nucleotides 1579 to 3788 of SEQ ID No. 1
correspond to the nucleotides 1 to 2201 of SEQ ID No.5 from WO
97/15664, but with a difference. During the production of SEQ ID
No.1 According to the invention, four additional nucleotides were
introduced, as a result of the cloning process, formed by a
reaction of filling an existing ECORI cutting site. This insertion
of the additional four nucleotides took place between nucleotide
357 and 358 of sequence SEQ ID No.5 from WO 97/15664. Nucleotides 1
to 1578 of the nucleic acid sequence with SEQ ID No. 1 from the
present invention, however, constitute new hitherto unknown
sequence regions which were isolated within the scope of this
invention. Also, WO 97/15664 did not disclose that SEQ ID No.1 from
the present invention or fragments or derivatives thereof increase
the transcription or expression of a gene of interest irrespective
of the chromosomal integration site when they are functionally
linked to a promoter/enhancer combination which allows the
transcription of the functionally linked gene of interest. Rather,
WO 97/15664 discloses the use of 5'UTR sequences of the
ubiquitin/S27a gene as promoter, while the sequence region from
position -161 to -45 according to FIG. 5 in WO 97/15664 is
essential for promoter activity. This sequence region is only
partly present in the nucleic acid of SEQ ID No. 1 according to the
invention and a fragment derived therefrom with SEQ ID No.2
(position -161 to -89 according to FIG. 5 in WO 97/15664). The
other fragments and derivatives of SEQ ID No.1 do not contain this
sequence region at all. Moreover, the nucleic acid of SEQ ID No. 1
according to the invention, when using standard alignment
algorithms such as BLAST, show no sequence homologies with the
nucleic acids sequences described in the following patent
applications, which can also positively influence the expression at
the chromatin level in cis: [0026] a) UCOE nucleic acid sequences
from WO 00/05393 [0027] b) EASE nucleic acid sequences from U.S.
Pat. No. 6,309,841 [0028] c) STAR nucleic acid sequences from WO
03/004704
DESCRIPTION OF THE FIGURES
[0029] FIG. 1: Schematic Representation of the Base Vectors
[0030] The vectors shown under A were used to express a recombinant
monoclonal IgG1 antibody in CHO-DG44 cells. "E/P" in this case is a
combination of CMV enhancer and hamster ubiquitin/S27a promoter,
"P" is merely a promoter element and "T" is a termination signal
for the transcription which is necessary for the polyadenylation of
the transcribed mRNA. The position and direction of transcription
initiation within each transcription unit is indicated by an arrow.
For cloning TE elements an SpeI cutting site ("SpeI") is present in
front of the promoter/enhancer combination. The amplifiable
selectable marker dihydrofolate reductase is abbreviated to "DHFR".
The selectable marker neomycin phosphotransferase contains the
point mutation D227G and is abbreviated to "D227G" accordingly in
the Figure. The "IRES" element originating from the
encephalomyocarditis virus acts as an internal ribosomal binding
site within the bicystronic transcription unit and allows
translation of the following green fluorescent protein "GFP". "HC"
and "LC" code for the heavy and light chains, respectively, of a
humanised monoclonal IgG1 antibody.
[0031] The vector shown under B was used to express the recombinant
protein MCP1 in CHO-DG44. "E/P" is a combination of CMV enhancer
and CMV promoter, "P" is merely a promoter element and "T" is a
termination signal for the transcription which is needed for the
polyadenylation of the transcribed mRNA. The position and direction
of transcription initiation within each transcription unit is
indicated by an arrow. For cloning the TE element, a sequence
region "A" with cutting sites for restriction endonucleases
(adapter) is inserted before the promoter.
[0032] The selectable marker neomycin phosphotransferase contains
the point mutation F240I and is accordingly abbreviated to F240I in
the figure. The IRES element originating from the
Encephalomyocarditis virus acts as an internal ribosomal binding
site within the bicistronic transcription unit and allows
translation of the subsequent red fluorescent protein "dsRed".
"MCP-1" codes for human monocyte chemoattractant Protein-1.
[0033] FIG. 2: Schematic Representation of an MCP-1 Base Vector
[0034] The vector shown here was used to express the recombinant
protein MCP-1 in CHO-DG44 cells. "E/P" is a combination of "E/P" is
a combination of CMV enhancer and CMV promoter, "P" is merely a
promoter element and "T" is a termination signal for the
transcription which is needed for the polyadenylation of the
transcribed mRNA. The position and direction of transcription
initiation within each transcription unit is indicated by an arrow.
For cloning the TE element, a sequence region "A" with cutting
sites for restriction endonucleases (adapter) is inserted before
the promoter. The selectable marker dihydrofolate reductase is
abbreviated to "dhfr" in the figure. The IRES element originating
from the Encephalomyocarditis virus acts as an internal ribosomal
binding site within the bicistronic transcription unit and allows
translation of the subsequent red fluorescent protein "dsRed".
"MCP-1" codes for human monocyte chemoattractant Protein-1.
[0035] FIG. 3: 5' Sequence of the CHO Ubiquitin/S27S Gene
[0036] The sequence region comprising 3788 by (SEQ ID No. 1) was
isolated from the genome of CHO (Chinese Hamster Ovary) cells and
is located upstream of the coding region of the Ub/S27a gene, which
is a fusion between a ubiquitin unit (Ub) and a ribosomal protein
of the small ribosome subunit (S27a).
[0037] FIG. 4: Graphic Representation of the TE Elements 00 to
12
[0038] This figure schematically shows the genomic sequence region
of 3788 bp, which was subcloned in a plasmid, located upstream of
the coding region of the CHO ubiquitin/S27a gene. From this genome
sequence (SEQ ID No. 1) also known as TE element A, partial
fragments of different lengths, hereinafter referred to as TE
elements, were prepared. TE element 00 (SEQ ID No. 2) was isolated
from a subclone of this sequence as a Sac II restriction fragment
and cloned into the SpeI cutting site of the target vectors pBID-HC
and pBING-LC. These contained either the gene for the heavy chain
(HC) or light chain of an IgG1 (see FIG. 1A). As a result,
expression vectors were formed in which the TE element 00 is
positioned in direct and reversed orientation upstream of the
promoter. The TE elements 01 to 12 were produced by PCR with
various pairs of primers (see FIGS. 5 and 6) and cloned into the
base plasmid pTE4/MCP-1 (FIG. 1B) and pTE5/MCP-1 (FIG. 2) via
BamHI/BsrGI.
[0039] FIG. 5: TE Elements 00 to 12
[0040] This Table shows the size and the starting and end positions
of the TE elements 00 to 21, which were produced from the TE-A
sequence (SEQ ID No. 1). For the fragments produced by PCR, the
primers used are additionally specified. The size gradations of the
elements are about 500 by and have deletions at the 5' or 3' end,
compared with the starting sequence TE-A (SEQ ID No.1).
[0041] FIG. 6: Primer for Synthesising the TE Elements 01 to 12
[0042] The primers are shown in the 5'-3' direction. Primers with
"for" in their name are primers in direct orientation of SEQ ID No.
1, primers with "rev" are those in reverse orientation. Each primer
consists at the 5' end of six nucleotides followed by a BamHI or
BsrGI cutting site and a sequence of about 20 to 30 nucleotides
which is 100% homologous with a sequence portion in SEQ ID No. 1.
The region of the primer homologous with SEQ ID No. 1 is shown in
bold. One for primer and one rev primer was used to amplify a
sequence region of SEQ ID No. 1. The resulting PCR product was
cloned into the base plasmid pTE4/MCP-1 (FIG. 1B) or pTE5/MCP-1
(FIG. 2) via BamHI and BsrGI.
[0043] FIG. 7: FACS Measurement of the Tranvection Series B
[0044] The Figure shows the relative increase in GFP expression in
cells with the TE element 00 compared with cells without the TE
element 00. For this, CHO-DG4 cells were transfected with the
plasmid combinations pBING-LC and pBID-HC, which differ from one
another only in the presence and orientation of the TE element 00.
After a two to three-week long selection of the transfected cell
pools in HT-free medium with the addition of G418, the GFP
fluorescence was measured by FACS analysis. Each graph, with the
exception of the untransfected CHO-DG44 cells (DG44) serving as a
negative control, constitutes the average of the GFP fluorescence
from, in each case, 10 pools of transfection series B. 20000 cells
were studied per pool. "Control" denotes the base plasmids pBING-LC
and pBID-HC, "reverse" denotes a reverse orientation of the TE
element 00 in the base vectors while "direct" indicates a direct
orientation of TE element 00 in the base vectors.
[0045] FIG. 8: FACS Measurement of Transvection Series C
[0046] The Figure shows the proportion of dsRed2-expressing cells
in stable cell populations which contained the TE elements 01, 02,
05, 06, 08 or 09, compared with cells in cell populations which did
not contain a TE element. For this, CHO-DG44 cells were transfected
with the plasmid pTE4/MCP-1 or derivatives obtained therefrom,
which additionally contained one of the TE elements mentioned
above. After an approximately three-week long selection of the
transfected cell pools in medium with added G418, the dsRed2
fluorescence was measured by FACS analysis. 10000 cells were
measured per pool and the inherent fluorescence of the
untransfected CHO-DG44 cells was substracted. Each value is the
average of the percentage proportion of dsRed2-expressing cells
from 6 pools of transfection series C.
[0047] FIG. 9: Influence of the TE Elements on the Specific
Productivity
[0048] This Figure shows the changes in the expression level of
IgG1 or MCP-1 which are obtained as a result of the presence of the
TE elements compared with control pools with no TE element, shown
graphically (A) or in table form (B). The cell pools were produced
by stable transfection of CHO-DG44 cells with the base plasmids
pBING-LC and pBID-HC or pTE4/MCP-1 ("control") and the derivatives
obtained therefrom, each of which additionally contained a TE
element ("00" in direct orientation ("00 direct") and in reverse
orientation ("00 reverse"), "01" to "12"). After a two to
three-week long selection of the transfected cell pools in HT-free
medium with the addition of G418 (Series A and B) or in
HT-containing medium with the addition of G418 (Series C and D) the
protein expression was measured by ELISA in the cell culture
supernatant and the specific productivity per cell and per day was
calculated. The cultivation of the stably transfected CHO-DG44
cells was carried out by several passages in 75 cm.sup.2 T flasks
with a passaging rythm of 2-2-3 days. In Series A, 4 pools taken
from the plasmid combinations "00 reverse" and "00 direct" were
tested and of the control 3 pools were tested over 8 passages in
culture, in Series B 10 pools of each plasmid combination were
tested by 6 passages and in Series C and D 6 pools of each type of
plasmid were tested through 6 passages. The specific productivities
of the pools of a plasmid combination and series were averaged and
the average of the controls in each series was set at 1. The
averaged specific productivities of the pools with TE element were
compared with this.
[0049] FIG. 10: Influence of the TE Elements on the Specific
Productivity in DHFR-Selected Cell Pools
[0050] This Figure shows the changes in the expression levels of
MCP-1 which resulted from the presence of the TE elements compared
with control pools with no TE element, in the form of a graph (A)
or table (B). The cell pools were produced by stable transfection
of CHO-DG44 cells with the base plasmid pTE5/MCP-1 ("control") or
derivatives obtained therefrom, each of which additionally
contained a TE element ("01" to "12") (Series E). After a two to
three-week long selection of the transfected cell pools in HT-free
medium the protein expression was measured by ELISA in the cell
culture supernatant and the specific productivity was calculated
per cell and per day. The cultivation of the stably transfected
CHO-DG44 cells was carried out by several passages in 75 cm.sup.2 T
flasks with a passaging rhythm of 2-2-3 days. Six pools of each
plasmid variant through 6 passages were in cultivation. The
specific productivities of the pools of a plasmid variant were
averaged and the average of the controls was set at 1. The averaged
specific productivities of the pools with a TE element was compared
with this.
[0051] FIG. 11: Testing the TE Elements for Enhancer Activity
[0052] The transient transfection of CHO-DG44 cells, when using
expression vectors with TE elements, showed no significant increase
in the MCP-1 titre compared with control vectors without a TE
element. TE elements 01 to 12 thus do not act as enhancers and can
therefore only bring about a significant increase in expression
when integrated in the chromosomes. Six pools were transfected with
pTE4/MCP-1 (control) and the derivatives obtained from it, which
additionally each contained a TE element ("01" to "12"). At the
same time an SEAP expression plasmid was co-transfected in order to
determine the transfection efficiency (SEAP=secreted alkaline
phosphatase). After 48 hours cultivation in a total volume of 3 ml,
the cell culture supernatant was removed and the MCP-1 titre was
determined by ELISA and the SEAP activity was determined. The MCP-1
titre was corrected with regard to the transfection efficiency,
determined by SEAP expression. The Figure shows the average of the
6 parallel pools with standard deviation.
[0053] FIG. 12: Other TE Elements
[0054] The results thus far indicate that the choice of fragments
of Sequence ID No. 1 shown in this Figure could also result in an
increase in gene expression. By cloning and stable transfection of
these additional TE elements the intention is to characterise
Sequence ID No. 1 more clearly in order to locate more precisely
the sequence regions which are important for the function.
[0055] FIG. 13: Testing of Different Positions and Combinations of
the TE Elements
[0056] This Figure represents a selection of possible expression
vectors in which different positions, orientation and combinations
of TE elements are used to investigate whether an additional
increase in expression can be achieved in this way. As well as the
flanking of the product gene by TE elements, a number of identical
or different short TE elements are also connected up one behind the
other, such as for example TE elements 06 and 08 or the new TE
elements 13 and 14.
[0057] FIG. 14: Influence of TE Elements TE 13 to TE 18 ON the
Specific MCP-1 Expression
[0058] This Figure graphically shows the changes in the expression
levels of MCP-1 which result from the presence of the TE elements
compared with control pools with no TE element. The cell pools are
produced by stable transfection of CHO-DG44 cells with the base
plasmid pTE4/MCP-1 ("control") or derivatives obtained therefrom
which additional each contained a TE element ("13" to "18") (Series
F). After a two to three-week long selection of the transfected
cell pools in HT-supplemented medium +G418 (400 .mu.g/ml) the
protein expression was measured by ELISA in the cell culture
supernatant and the specific productivity per cell and per day was
calculated. Cultivation of the stably transfected CHO-DG44 cells
was carried out by several passages in 75 cm.sup.2 T flasks with a
passaging rhythm of 2-2-3 days. Of each plasmid variant, 4 pools
were in cultivation over 5 to 6 passages. The specific
productivities of the pools of a plasmid variant were averaged and
the average of the controls was set at 1. The averaged specific
productivities of the pools with a TE element were compared with
this.
[0059] FIG. 15: Influence of the TE Elements at Various Positions
and in Various Combinations on the Expression of MCP-1
[0060] This Figure graphically shows the changes in the expression
levels of MCP-1 which result from the presence and combination of
different TE elements compared with control pools without a TE
element. The cell pools were produced by stable transfection
CHO-DG44 cells with the base plasmid pTE4/MCP-1 ("control") or
derivatives obtained therefrom which additionally each contained
one or two TE elements ("06 and 08, 08rev, 09rev, A") (Series G).
After two to three-week long selection of the transfected cell
pools in HT-supplemented medium +G418 (300 .mu.g/ml) the protein
expression was measured by ELISA in the cell culture supernatant
and the specific productivity per cell and per day was calculated.
The cultivation of the stably transfected CHO-DG44 cells was
carried out by several passages in 6-well plates (MATE) with a
passaging rhythm of 2-2-3 days. Of each plasmid variant, 6 pools
were in cultivation over 6 passages. The specific productivities of
the pools of a plasmid variant were averaged and the average value
of the controls was set at 1. The averaged specific productivities
of the pools with a TE element were compared with this.
[0061] FIG. 16: Testing of the TE Element TE-08 with IgG-4
Antibodies
[0062] The vectors shown here were used for the expression of
recombinant monoclonal IgG4 antibodies in CHO-DG44 cells. E/P in
this case is a combination of CMV enhancer and promoter, P is
merely a promoter element and T is a termination signal for the
transcription, which is required for the polyadenylation of the
transcribed mRNA. The position and direction of the transcription
initiation within each transcription unit is indicated by an arrow.
The genes for the light chain (LC) and heavy chain (HC) were cloned
in, in exchange for the MCP-1--IRES--dsRed2 cassette (FIGS. 1B and
2). The code for the heavy and light chains, respectively, of a
humanise monoclonal IgG-4 antibody. The amplifiable selectable
marker dihydrofolate reductase is abbreviated to "dhfr". The
selectable marker neomycin-phosphotransferase contains the point
mutation F240I and is abbreviated accordingly to F240I in the
Figure.
DETAILED DESCRIPTION OF THE INVENTION
[0063] Terms and designations used within the scope of this
description of the invention have the following meanings defined
hereinafter. The general terms "containing" or "contains" includes
the more specific term "consisting of". Moreover, the terms "single
number" and "plurality" are not used restrictively.
[0064] The term "TE element" denotes regulatory nucleic acids.
[0065] The terms "TE element" or "expression-enhancing element" or
"transcription enhancing element" or "expression or transcription
enhancing nucleic acid element" are used synonymously in the test.
These terms all refer to regulatory nucleic acid sequences.
[0066] By "TE element" or "expression enhancing element" or
"transcription enhancing element" or "expression or transcription
enhancing nucleic acid element" is meant in particular Sequence ID
No. 1, including the complementary sequence thereto, which was
isolated from the genome of the Chinese hamster (Cricetulus
griseus), or any part, fragment or region thereof or a derivative
of Sequence ID No. 1 or one of the parts, fragments or regions
thereof, which when stably integrated in the chromosomes leads to
an increase in the transcription or expression of a gene of
interest. Also meant are any desired combinations of parts,
fragments, regions or derivatives of Sequence ID No. 1 which
consist of a number of identical or different parts, fragments,
regions or derivatives of SEQ ID No. 1, which may in turn be
arranged in any desired orientation and at any desired spacing
relative to one another or may be combined with other regulatory
sequences and which lead to an increase in the transcription or
expression of a gene of interest. The term TE element may refer
both to SEQ ID No. 1 itself and to any desired fragments, parts,
regions or derivatives thereof.
[0067] Furthermore, the term "TE element", "transcription enhancing
or expression enhancing nucleic acid element" or fragments, parts,
regions or derivatives thereof encompasses, in addition to parts of
the sequence of the Chinese hamster (Cricetulus griseus),
corresponding functional homologous nucleotide sequences from other
organisms. Examples of these other organisms include man, mouse,
rat, monkey and other mammals and rodents, reptiles, birds, fishes
and plants.
[0068] By a "fragment" or "part" or "region" (these terms being
used synonymously) is meant a nucleic acid molecule (single or
double stranded) which is 100% identical in its sequence to a part
of SEQ ID No. 1 or the complementary sequence thereto. It is known
that the cloning of fragments which are produced either by
digestion with restriction enzymes or by PCR can lead to
modifications in the end regions of the fragment, i.e. additional
or absent nucleotides or nucleotides additionally introduced
through primers, which are the result of filling or breakdown
reactions. These variations in the end regions of the fragments are
included in the definition of a fragment, even if these sequence
regions have a sequence identity of less than 100% with SEQ ID No.
1. "Parts" or "fragments" or "regions" of Sequence ID No. 1 include
for example TE-00 (Sequence ID No. 2), TE-01 (Sequence ID No. 3),
TE-02 (Sequence ID No. 4), TE-03 (Sequence ID No. 5), TE-04
(Sequence ID No. 6), TE-05 (Sequence ID No. 7), TE-06 (Sequence ID
No. 8), TE-07 (Sequence ID No. 9), TE-08 (Sequence ID No. 10),
TE-09 (Sequence ID No. 11), TE-10 (Sequence ID No. 12), TE-11
(Sequence ID No. 13), TE-12 (Sequence ID No. 14), TE-13 (Sequence
ID No. 15), TE-14 (Sequence ID No. 16), TE-15 (Sequence ID No. 17),
TE-16 (Sequence ID No. 18), TE-17 (Sequence ID No. 19), TE-18
(Sequence ID No. 20). Preferably, with stable chromosomal
integration, the fragment leads to an increase in the transcription
or is expression of a functionally linked gene of interest. "Parts"
or "fragments" or "regions" of Sequence ID No. 1, which lead to an
increase in the transcription or expression of a gene of interest,
are for example TE-00 (Sequence ID No. 2), TE-01 (Sequence ID No.
3), TE-02 (Sequence ID No. 4), TE-03 (Sequence ID No. 5), TE-04
(Sequence ID No. 6), TE-06 (Sequence ID No. 8), TE-07 (Sequence ID
No. 9), TE-08 (Sequence ID No. 10), TE-10 (Sequence ID No. 12),
TE-11 (Sequence ID No. 13), TE-12 (Sequence ID No. 14). However,
the term "fragment" also includes all possible other parts of SEQ
ID No. 1 in any desired orientation which lead to an increase in
the transcription or expression of a gene of interest, particularly
those which are wholly or at least partially in the 5' region of
TE-00 (SEQ ID No. 2). This corresponds to the partial region of SEQ
ID No. 1 between 1 by and 1578 bp. Also preferred is the fragment
TE-08 (SEQ ID No. 10).
[0069] By a "derivative" is meant, in the present invention, a
nucleic acid molecule (single or double stranded) which has at
least 70% sequence identity, preferably at least about 80% sequence
identity, particularly preferably at least about 90% sequence
identity and most preferably at least about 95% sequence identity
with SEQ ID No. 1 or the complementary sequence thereto or with a
part or fragment or region of SEQ ID No. 1 or the complementary
sequence thereto, and which, on chromosomal integration, leads to
an increase in the transcription or expression of a gene of
interest. Sequence differences from SEQ ID No. 1 may be based on
the one hand on differences in homologous endogenous nucleotide
sequences from other organisms. On the other hand they may also be
based on deliberate modifications of the nucleotide acid sequence,
e.g. on substitution, insertion or deletion of at least one or more
nucleotides. Deletion, insertion and substitution mutants can be
produced by "site-specific mutagenesis" and/or "PCR-based
mutagenesis techniques". Corresponding methods are described by way
of example by Lottspeich and Zorbas (1998; Chapter 36.1 with
further references). The sequence identity can be brought into
conformity with a reference sequence, in this case Sequence ID No.
1, using so-called standard alignment algorithms such as for
example "BLAST" (Altschul, S. F., Gish, W., Miller, W., Myers, E.
W. & Lipman, D. J. (1990) "Basic local alignment search tool."
J. Mol. Biol. 215:403-410; Madden, T. L., Tatusov, R. L. &
Zhang, J. (1996) "Applications of network BLAST server" Meth.
Enzymol. 266:131-141; Zhang, J. & Madden, T. L. (1997)
"PowerBLAST: A new network BLAST application for interactive or
automated sequence analysis and annotation." Genome Res.
7:649-656). Sequences are brought into conformity when they
correspond in their succession and can be identified using standard
alignment algorithms
[0070] By a "derivative" is meant, according to the present
invention, a nucleic acid molecule (single or double stranded)
which hybridises with SEQ ID No. 1 or with the sequence of a
fragment or part or region of SEQ ID No. 1 or of a sequence
complementary thereto. Preferably the hybridisation is carried out
under stringent hybridisation and washing conditions (e.g.
hybridisation at 65.degree. C. in a buffer containing 5.times.SSC;
washing at 42.degree. C. with 0.2.times.SSC/0.1% SDS).
Corresponding techniques are described by way of example in Ausubel
et al., 1994. Preferably the part or fragment or region of SEQ ID
No. 1 includes all or at least parts of the sequence region between
nucleotide position 1 by and 1578 bp. This corresponds to sequence
region 5' of the TE-00 sequence (SEQ ID No. 2). The fragment TE-08
(SEQ ID No. 10) is also preferred.
[0071] The term "variant" refers to the expression vectors used in
the particular transfection mixture. These include both the base
vectors (pTE4/MCP-1 or pTE5/MCP-1) or the base vector combination
(pBING-LC+pBID-HC) and also the base vectors which contain one or
more TE elements in different positions, combinations and
orientations.
[0072] In the case of primers, the term "orientation" refers to the
arrangement of the primers in relation to SEQ ID No. 1. All the
primers whose sequence order corresponds to the sequence in the
5'-3' order shown under SEQ ID No.1 (=forward primer) are in the
same orientation as this sequence, which is also referred to as
"direct orientation". Primers whose sequence order is complementary
to the sequence given under SEQ ID No.1 (=reverse primer) are in
the opposite orientation to this sequence, which is also referred
to as "reverse orientation". In connection with TE elements, in the
present invention, the term "orientation" refers to the arrangement
in relation to the gene of interest. The sequence given under SEQ
ID No. 1 represents a genome sequence which is positioned 5' from
the region coding for the ubiquitin/S27a gene, which is also
referred to as "upstream".
[0073] The continuation of this sequence shown in SEQ ID No.1 in
the direction of the coding region of the following ubiquitin/S27a
gene would lead to the start codon of this gene. This arrangement
is therefore referred to as "direct orientation". Analogously, in
the present invention, the TE element is in the direct orientation
when the sequence shown in SEQ ID No.1, or any desired part,
fragment, region or derivative thereof, is present in the
expression vector on the same DNA strand as the start codon of the
gene of interest. If, by contrast, the sequence complementary to
SEQ ID No.1, or any desired part, fragment, region or derivative
thereof, is present in the expression vector on the same DNA strand
as the start codon of the gene of interest, then the TE element is
in a "reverse orientation". Unless stated otherwise, when a TE
element is mentioned, both orientations are always included/meant,
i.e. both direct and reverse.
[0074] By "chromosomal integration" is meant the integration of any
desired nucleic acid sequence into the genome, i.e. into the
chromosomes, of a cell, this integration optionally being into one
or more chromosomes in any desired number, position and
orientation. Moreover, the term "chromosomal integration" also
includes the integration of any desired nucleic acid sequence into
synthetic, artificial or mini-chromosomes.
Gene of Interest:
[0075] The gene of interest contained in the expression vector
according to the invention comprises a nucleotide sequence of any
length which codes for a product of interest. The gene product or
"product of interest" is generally a protein, polypeptide, peptide
or fragment or derivative thereof. However, it may also be RNA or
antisense RNA. The gene of interest may be present in its full
length, in shortened form, as a fusion gene or as a labelled gene.
It may be genomic DNA or preferably cDNA or corresponding fragments
or fusions. The gene of interest may be the native gene sequence,
or it may be mutated or otherwise modified. Such modifications
include codon optimisations for adapting to a particular host cell
and humanisation. The gene of interest may, for example, code for a
secreted, cytoplasmic, nuclear-located, membrane-bound or cell
surface-bound polypeptide.
[0076] The term "nucleotide sequence", "nucleotide sequence" or
"nucleic acid sequence" indicates an oligonucleotide, nucleotides,
polynucleotides and fragments thereof as well as DNA or RNA of
genomic or synthetic origin which occur as single or double strands
and can represent the coding or non-coding strand of a gene.
Nucleic acid sequences may be modified using standard techniques
such as site-specific mutagenesis or PCR-mediated mutagenesis (e.g.
described in Sambrook et al., 1989 or Ausubel et al., 1994).
[0077] By "coding" is meant the property or capacity of a specific
sequence of nucleotides in a nucleic acid, for example a gene in a
chromosome or an mRNA, to act as a matrix for the synthesis of
other polymers and macromolecules such as for example rRNA, tRNA,
mRNA, other RNA molecules, cDNA or polypeptides in a biological
process. Accordingly, a gene codes for a protein if the desired
protein is produced in a cell or another biological system by
transcription and subsequent translation of the mRNA. Both the
coding strand whose nucleotide sequence is identical to the mRNA
sequence and is normally also given in sequence databanks, e.g.
EMBL or GenBank, and also the non-coding strand of a gene or cDNA
which acts as the matrix for transcription may be referred to as
coding for a product or protein. A nucleic acid which codes for a
protein also includes nucleic acids which have a different order of
nucleotide sequence on the basis of the degenerate genetic code but
result in the same amino acid sequence of the protein. Nucleic acid
sequences which code for proteins may also contain introns.
[0078] The term "cDNA" denotes deoxyribonucleic acids which are
prepared by reverse transcription and synthesis of the second DNA
strand from a mRNA or other RNA produced from a gene. If the cDNA
is present as a double stranded DNA molecule it contains both a
coding and a non-coding strand.
[0079] The term "intron" denotes non-coding nucleotide sequences of
any length. They occur naturally in numerous eukaryotic genes and
are eliminated from a previously transcribed mRNA precursor by a
process known as splicing. This requires precise excision of the
intron at the 5' and 3' ends and correct joining of the resulting
mRNA ends so as to produce a mature processed mRNA with the correct
reading frame for successful protein synthesis. Many of the splice
donor and splice acceptor sites involved in this splicing process,
i.e. the sequences located directly at the exon-intron or
intron-exon interfaces, have been characterised by now. For an
overview see Ohshima et al., 1987.
Protein/Product of Interest
[0080] Proteins/polypeptides with a biopharmaceutical significance
include for example antibodies, enzymes, cytokines, lymphokines,
adhesion molecules, receptors and the derivatives or fragments
thereof, but are not restricted thereto. Generally, all
polypeptides which act as agonists or antagonists and/or have
therapeutic or diagnostic applications may be used. Other proteins
of interest are, for example, proteins/polypeptides, which are used
to change the properties of host cells within the scope of
so-called "Cell Engineering", such as e.g. anti-apoptotic proteins,
chaperones, metabolic enzymes, glycosylation enzymes and the
derivatives or fragments thereof, but are not restricted
thereto.
[0081] The term "polypeptides" is used for amino acid sequences or
proteins and refers to polymers of amino acids of any length. This
term also includes proteins which have been modified
post-translationally by reactions such as glycosylation,
phosphorylation, acetylation or protein processing. The structure
of the polypeptide may be modified, for example, by substitutions,
deletions or insertions of amino acids and fusion with other
proteins while retaining its biological activity. In addition, the
polypeptides may multimerise and form homo- and heteromers.
[0082] Examples of therapeutic proteins are insulin, insulin-like
growth factor, human growth hormone (hGH) and other growth factors,
receptors, tissue plasminogen activator (tPA), erythropoietin
(EPO), cytokines, e.g. interleukines (IL) such as IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,
IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN)-alpha, -beta,
-gamma, -omega or -tau, tumour necrosis factor (TNF) such as
TNF-alpha, beta or gamma, TRAIL, G-CSF, GM-CSF, M-CSF, MCP-1 and
VEGF. Other examples are monoclonal, polyclonal, multispecific and
single chain antibodies and fragments thereof such as for example
Fab, Fab', F(ab').sub.2, Fc and Fc' fragments, light (L) and heavy
(H) immunoglobulin chains and the constant, variable or
hypervariable regions thereof as well as Fv and Fd fragments
(Chamov et al., 1999). The antibodies may be of human or non-human
origin. Humanised and chimeric antibodies are also possible.
[0083] Fab fragments (fragment antigen binding=Fab) consist of the
variable regions of both chains which are held together by the
adjacent constant regions. They may be produced for example from
conventional antibodies by treating with a protease such as papain
or by DNA cloning. Other antibody fragments are F(ab').sub.2
fragments which can be produced by proteolytic digestion with
pepsin.
[0084] By gene cloning it is also possible to prepare shortened
antibody fragments which consist only of the variable regions of
the heavy (VH) and light chain (VL). These are known as Fv
fragments (fragment variable=fragment of the variable part). As
covalent binding via the cysteine groups of the constant chains is
not possible in these Fv fragments, they are often stabilised by
some other method. For this purpose the variable regions of the
heavy and light chains are often joined together by means of a
short peptide fragment of about 10 to 30 amino acids, preferably 15
amino acids. This produces a single polypeptide chain in which VH
and VL are joined together by a peptide linker. Such antibody
fragments are also referred to as single chain Fv fragments (scFv).
Examples of scFv antibodies are known and described, cf. for
example Huston et al., 1988.
[0085] In past years various strategies have been developed for
producing multimeric scFv derivatives. The intention is to produce
recombinant antibodies with improved pharmacokinetic properties and
increased binding avidity. In order to achieve the multimerisation
of the scFv fragments they are produced as fusion proteins with
multimerisation domains. The multimerisation domains may be, for
example, the CH3 region of an IgG or helix structures ("coiled coil
structures") such as the Leucine Zipper domains. In other
strategies the interactions between the VH and VL regions of the
scFv fragment are used for multimerisation (e.g. dia, tri- and
pentabodies).
[0086] The term "diabody" is used in the art to denote a bivalent
homodimeric scFv derivative. Shortening the peptide linker in the
scFv molecule to 5 to 10 amino acids results in the formation of
homodimers by superimposing VH/VL chains. The diabodies may
additionally be stabilised by inserted disulphite bridges. Examples
of diabodies can be found in the literature, e.g. in Perisic et
al., 1994.
[0087] The term "minibody" is used in the art to denote a bivalent
homodimeric scFv derivative. It consists of a fusion protein which
contains the CH3 region of an immunoglobulin, preferably IgG, most
preferably IgG1, as dimerisation region. This connects the scFv
fragments by means of a hinge region, also of IgG, and a linker
region. Examples of such minibodies are described by Hu et al.,
1996.
[0088] The term "triabody" is used in the art to denote a trivalent
homotrimeric scFv derivative (Kortt et al., 1997). The direct
fusion of VH-VL without the use of a linker sequence leads to the
formation of trimers.
[0089] The fragments known in the art as mini antibodies which have
a bi, tri- or tetravalent structure are also derivatives of scFv
fragments. The multimerisation is achieved by means of di-, tri- or
tetrameric coiled coil structures (Pack et al., 1993 and 1995;
Lovejoy et al., 1993).
Gene which Codes for a Fluorescent Protein:
[0090] In another embodiment the expression vector according to the
invention contains a gene coding for a fluorescent protein,
functionally linked to the gene of interest. Preferably, both genes
are transcribed under the control of a single heterologous promoter
so that the protein/product of interest and the fluorescent protein
are coded by a bicistronic mRNA. This makes it possible to identify
cells which produce the protein/product of interest in large
amounts, by means of the expression rate of the fluorescent
protein. Alternatively, the transcription of the gene coding for
the fluorescent protein may take place under the control of its own
promoter.
[0091] The fluorescent protein may be, for example, a green,
bluish-green, blue, yellow or other coloured fluorescent protein.
One particular example is green fluorescent protein (GFP) obtained
from Aequorea victoria or Renilla reniformis and mutants developed
from them; cf. for example Bennet et al., 1998; Chalfie et al.,
1994; WO 01/04306 and the literature cited therein.
[0092] Other fluorescent proteins and genes coding for them are
described in WO 00/34318, WO 00/34326, WO 00/34526 and WO 01/27150
which are incorporated herein by reference. These fluorescent
proteins are fluorophores of non-bioluminescent organisms of the
species Anthozoa, for example Anemonia majano, Clavularia sp.,
Zoanthus sp. I, Zoanthus sp. II, Discosoma striata, Discosoma sp.
"red", Discosoma sp. "green", Discosoma sp. "Magenta", Anemonia
sulcata, Aequorea coerulescens.
[0093] The fluorescent proteins used according to the invention
contain in addition to the wild-type proteins natural or
genetically engineered mutants and variants, fragments, derivatives
or variants thereof which have for example been fused with other
proteins or peptides. The mutations used may for example alter the
excitation or emission spectrum, the formation of chromophores, the
extinction coefficient or the stability of the protein. Moreover,
the expression in mammalian cells or other species can be improved
by codon optimisation. According to the invention the fluorescent
protein may also be used in fusion with a selectable marker,
preferably an amplifiable selectable marker such as dihydrofolate
reductase (DHFR).
[0094] The fluorescence emitted by the fluorescent proteins makes
it possible to detect the proteins, e.g. by throughflow cytometry
with a fluorescence-activated cell sorter (FACS) or by fluorescence
microscopy.
Other Regulatory Elements:
[0095] The expression vector contains at least one heterologous
promoter which allows expression of the gene of interest and
preferably also of the fluorescent protein.
[0096] The term "promoter" denotes a polynucleotide sequence which
allows and controls the transcription of the genes or sequences
functionally connected therewith. A promoter contains recognition
sequences for binding RNA polymerase and the initiation site for
transcription (transcription initiation site). In order to express
a desired sequence in a certain cell type or a host cell a suitable
functional promoter must be chosen. The skilled man will be
familiar with a variety of promoters from various sources,
including constitutive, inducible and repressible promoters. They
are deposited in databanks such as GenBank, for example, and may be
obtained as separate elements or elements cloned within
polynucleotide sequences from commercial or individual sources. In
inducible promoters the activity of the promoter may be reduced or
increased in response to a signal. One example of an inducible
promoter is the tetracycline (tet) promoter. This contains
tetracycline operator sequences (tetO) which can be induced by a
tetracycline-regulated transactivator protein (tTA). In the
presence of tetracycline the binding of tTA to tetO is inhibited.
Examples of other inducible promoters are the jun, fos,
metallothionein and heat shock promoter (see also Sambrook et al.,
1989; Gossen et al., 1994).
[0097] Of the promoters which are particularly suitable for high
expression in eukaryotes, there are for example the ubiquitin/S27a
promoter of the hamster (WO 97/15664), SV 40 early promoter,
adenovirus major late promoter, mouse metallothionein-I promoter,
the long terminal repeat region of Rous Sarcoma Virus, the early
promoter of human Cytomegalovirus. Examples of other heterologous
mammalian promoters are the actin, immunoglobulin or heat shock
promoter(s).
[0098] A corresponding heterologous promoter can be functionally
connected to other regulatory sequences in order to
increase/regulate the transcription activity in an expression
cassette.
[0099] For example, the promoter may be functionally linked to
enhancer sequences in order to increase the transcriptional
activity. For this, one or more enhancers and/or several copies of
an enhancer sequence may be used, e.g. a CMV or SV40 enhancer.
Accordingly, an expression vector according to the invention, in
another embodiment, contains one or more enhancers/enhancer
sequences, preferably a CMV or SV40 enhancer.
[0100] The term enhancer denotes a polynucleotide sequence which in
the cis location acts on the activity of a promoter and thus
stimulates the transcription of a gene functionally connected to
this promoter. Unlike promoters the effect of enhancers is
independent of position and orientation and they can therefore be
positioned in front of or behind a transcription unit, within an
intron or even within the coding region. The enhancer may be
located both in the immediate vicinity of the transcription unit
and at a considerable distance from the promoter. It is also
possible to have a physical and functional overlap with the
promoter. The skilled man will be aware of a number of enhancers
from various sources (and deposited in databanks such as GenBank,
e.g. SV40 enhancers, CMV enhancers, polyoma enhancers, adenovirus
enhancers) which are available as independent elements or elements
cloned within polynucleotide sequences (e.g. deposited at the ATCC
or from commercial and individual sources). A number of promoter
sequences also contain enhancer sequences such as the frequently
used CMV promoter. The human CMV enhancer is one of the strongest
enhancers identified hitherto. One example of an inducible enhancer
is the metallothionein enhancer, which can be stimulated by
glucocorticoids or heavy metals.
[0101] Another possible modification is, for example, the
introduction of multiple Sp 1 binding sites. The promoter sequences
may also be combined with regulatory sequences which allow
control/regulation of the transcription activity. Thus, the
promoter can be made repressible/inducible. This can be done for
example by linking to sequences which are binding sites for up- or
down-regulating transcription factors. The above mentioned
transcription factor Sp 1, for example, has a positive effect on
the transcription activity. Another example is the binding site for
the activator protein AP1, which may act both positively and
negatively on transcription. The activity of AP1 can be controlled
by all kinds of factors such as, for example, growth factors,
cytokines and serum (Faisst et al., 1992 and references therein).
The transcription efficiency can also be increased by changing the
promoter sequence by the mutation (substitution, insertion or
deletion) of one, two, three or more bases and then determining, in
a reporter gene assay, whether this has increased the promoter
activity.
[0102] Basically, the additional regulatory elements include
heterologous promoters, enhancers, termination and polyadenylation
signals and other expression control elements. Both inducible and
constitutively regulatory sequences are known for the various cell
types.
[0103] "Transcription-regulatory elements" generally comprise a
promoter upstream of the gene sequence to be expressed,
transcription initiation and termination sites and a
polyadenylation signal.
[0104] The term "transcription initiation site" refers to a nucleic
acid in the construct which corresponds to the first nucleic acid
which is incorporated in the primary transcript, i.e. the mRNA
precursor. The transcription initiation site may overlap with the
promoter sequences.
[0105] The term "transcription termination site" refers to a
nucleotide sequence which is normally at the 3' end of the gene of
interest or of the gene section which is to be transcribed, and
which brings about the termination of transcription by RNA
polymerase.
[0106] The "polyadenylation signal" is a signal sequence which
causes cleavage at a specific site at the 3' end of the eukaryotic
mRNA and post-transcriptional incorporation of a sequence of about
100-200 adenine nucleotides (polyA tail) at the cleaved 3' end. The
polyadenylation signal comprises the sequence AATAAA about 10-30
nucleotides upstream of the cleavage site and a sequence located
downstream. Various polyadenylation elements are known such as tk
polyA, SV40 late and early polyA or BGH polyA (described for
example in U.S. Pat. No. 5,122,458).
[0107] "Translation regulatory elements" comprise a translation
initiation site (AUG), a stop codon and a polyA signal for each
polypeptide to be expressed. For optimum expression it may be
advisable to remove, add or change 5'- and/or 3'-untranslated
regions of the nucleic acid sequence which is to be expressed, in
order to eliminate any potentially unsuitable additional
translation initiation codons or other sequences which might affect
expression at the transcription or expression level. In order to
promote expression, ribosomal consensus binding sites may
alternatively be inserted immediately upstream of the start codon.
In order to produce a secreted polypeptide the gene of interest
usually contains a signal sequence which codes for a signal
precursor peptide which transports the synthesised polypeptide to
and through the ER membrane. The signal sequence is often but not
always located at the amino terminus of the secreted protein and is
cleaved by signal peptidases after the protein has been filtered
through the ER membrane. The gene sequence will usually but not
necessarily contain its own signal sequence. If the native signal
sequence is not present a heterologous signal sequence may be
introduced in known manner. Numerous signal sequences of this kind
are known to the skilled man and deposited in sequence databanks
such as GenBank and EMBL.
[0108] Another regulatory element is the internal ribosomal entry
site (IRES). The IRES element comprises a sequence which
functionally activates the translation initiation independently of
a 5'-terminal methylguanosinium cap (CAP structure) and the
upstream gene and in an animal cell allows the translation of two
cistrons (open reading frames) from a single transcript. The IRES
element provides an independent ribosomal entry site for the
translation of the open reading frame located immediately
downstream. In contrast to bacterial mRNA which may be
multicistronic, i.e. it may code for numerous different
polypeptides or products which are translated one after the other
by the mRNA, the majority of mRNAs from animal cells are
monocistronic and code for only one protein or product. In the case
of a multicistronic transcript in a eukaryotic cell the translation
would be initiated from the translation initiation site which was
closest upstream and would be stopped by the first stop codon,
after which the transcript would be released from the ribosome.
Thus, only the first polypeptide or product coded by the mRNA would
be produced during translation. By contrast, a multicistronic
transcript with an IRES element which is functionally linked to the
second or subsequent open reading frame in the transcript allows
subsequent translation of the open reading frame located downstream
thereof, so that two or more polypeptides or products coded by the
same transcript are produced in the eukaryotic cell.
[0109] The IRES element may be of various lengths and various
origins and may originate, for example, from the
encephalomyocarditis virus (EMCV) or other Picorna viruses. Various
IRES sequences and their use in the construction of vectors are
described in the literature, cf. for example Pelletier et al.,
1988; hug et al., 1989; Davies et al., 1992; Adam et al., 1991;
Morgan et al., 1992; Sugimoto et al., 1994; Ramesh et al., 1996;
Mosser et al., 1997.
[0110] The gene sequence located downstream is functionally linked
to the 3' end of the IRES element, i.e. the spacing is selected so
that the expression of the gene is unaffected or only marginally
affected or has sufficient expression for the intended purpose. The
optimum permissible distance between the IRES element and the start
codon of the gene located downstream thereof for sufficient
expression can be determined by simple experiments by varying the
spacing and determining the expression rate as a function of the
spacing using reporter gene assays.
[0111] By the measures described it is possible to obtain an
optimum expression cassette which is of great value for the
expression of heterologous gene products. An expression cassette
obtained by means of one or more such measures is therefore a
further subject of the invention.
Hamster-Ubiquitin/S27a Promoter:
[0112] In another embodiment the expression vector according to the
invention contains the ubiquitin/S27a promoter of the hamster,
preferably functionally linked to the gene of interest and even
more preferably functionally linked to the gene of interest and the
gene which codes for a fluorescent protein or a selectable
marker.
[0113] The ubiquitin/S27a promoter of the hamster is a powerful
homologous promoter which is described in WO 97/15664. Such a
promoter preferably has at least one of the following features:
GC-rich sequence area, Sp 1 binding site, polypyrimidine element,
absence of a TATA box. Particularly preferred is a promoter which
has an Sp 1 binding site but no TATA box. Also preferred is a
promoter which is constitutively activated and in particular is
equally active under serum-containing, low-serum and serum-free
cell culture conditions. In another embodiment it is an inducible
promoter, particularly a promoter which is activated by the removal
of serum.
[0114] A particularly advantageous embodiment is a promoter with a
nucleotide sequence as contained in FIG. 5 of WO 97/15664.
Particularly preferred are promoter sequences which contain the
sequence from position -161 to -45 of FIG. 5.
[0115] The promoters used in the examples of the present patent
specification each contain a DNA molecule with a sequence which
corresponds to the fragment -372 to +111 from FIG. 5 of WO 97/15664
and represents the preferred promoter, i.e a preferred promoter
should incorporate this sequence region.
Preparation of Expression Vectors According to the Invention:
[0116] The expression vector according to the invention may
theoretically be prepared by conventional methods known in the art,
as described by Sambrook et al. (1989), for example. Sambrook also
describes the functional components of a vector, e.g. suitable
promoters (in addition to the hamster ubiquitin/S27a promoter),
enhancers, termination and polyadenylation signals, antibiotic
resistance genes, selectable markers, replication starting points
and splicing signals. Conventional cloning vectors may be used to
produce them, e.g. plasmids, bacteriophages, phagemids, cosmids or
viral vectors such as baculovirus, retroviruses, adenoviruses,
adeno-associated viruses and herpes simplex virus, as well as
synthetic or artificial chromosomes/mini chromosomes. The
eukaryotic expression vectors typically also contain prokaryotic
sequences such as, for example, replication origin and antibiotic
resistance genes which allow replication and selection of the
vector in bacteria. A number of eukaryotic expression vectors which
contain multiple cloning sites for the introduction of a
polynucleotide sequence are known and some may be obtained
commercially from various companies such as Stratagene, La Jolla,
Calif., USA; Invitrogen, Carlsbad, Calif., USA; Promega, Madison,
Wis., USA or BD Biosciences Clontech, Palo Alto, Calif., USA.
[0117] The heterologous promoter, the gene (or genes) of interest,
selectable markers and optionally the gene coding for a fluorescent
protein, additional regulatory elements such asthe internal
ribosomal entry site (IRES), enhancers, a polyadenylation signal
and other cis-active elements such as TE elements, for example, are
introduced into the expression vector in a manner familiar to those
skilled in the art. An expression vector according to the invention
contains, at the minimum, a heterologous promoter, the gene of
interest and a TE element. Preferably, the expression vector also
contains a gene coding for a fluorescent protein. It is
particularly preferred according to the invention to use a
ubiquitin/S27a promoter as heterologous promoter. Particularly
preferred is an expression vector in which the heterologous
promoter, preferably a ubiquitin/S27a promoter, the gene of
interest and a TE element are functionally linked together or are
functionally linked.
[0118] Within the scope of the present description the term
"functional linking" or "functionally linked" refers to two or more
nucleic acid sequences or partial sequences which are positioned so
that they can perform their intended function. For example, a
promoter/enhancer, a promoter/TE element or a promoter/enhancer/TE
element is functionally linked to a coding gene sequence if it is
able to control or modulate the transcription of the linked gene
sequence in the cis position. Generally, but not necessarily,
functionally linked DNA sequences are close together and, if two
coding gene sequences are linked or in the case of a secretion
signal sequence, in the same reading frame. Although a functionally
linked promoter is generally located upstream of the coding gene
sequence it does not necessarily have to be close to it. Enhancers
need not be close by either, provided that they assist the
transcription or expression of the gene sequence. For this purpose
they may be both upstream and downstream of the gene sequence,
possibly at some distance from it. A polyadenylation site is
functionally linked to a gene sequence if it is positioned at the
3' end of the gene sequence in such a way that the transcription
progresses via the coding sequence to the polyadenylation signal.
Linking may take place according to conventional recombinant
methods, e.g. by the PCR technique, by ligation at suitable
restriction cutting sites or by splicing. If no suitable
restriction cutting sites are available synthetic oligonucleotide
linkers or adaptors may be used in a manner known per se.
[0119] In one of the embodiments described, the heterologous
promoter, preferably a ubiquitin/S27a promoter or CMV promoter, the
gene of interest and the gene coding for a fluorescent protein are
functionally linked together. This means for example that both the
gene of interest and the gene coding for a fluorescent protein are
expressed starting from the same heterologous promoter. In a
particularly preferred embodiment the functional linking takes
place via an IRES element, so that a bicistronic mRNA is
synthesised from both genes. The expression vector according to the
invention may additionally contain enhancer elements and/or TE
elements which act functionally on one or more promoters.
Particularly preferred is an expression vector in which the
heterologous promoter, preferably the ubiquitin/S27a promoter or a
modified form thereof or the CMV promoter, is linked to an enhancer
element, e.g. an SV40 enhancer or a CMV enhancer element, and a TE
element.
[0120] Fundamentally, the expression of the genes within an
expression vector may take place starting from one or more
transcription units. The term transcription unit is defined as a
region which contains one or more genes to be transcribed. The
genes within a transcription unit are functionally linked to one
another in such a way that all the genes within such a unit are
under the transcriptional control of the same promoter,
promoter/enhancer or promoter/enhancer/TE element. As a result of
this transcriptional linking of genes, more than one protein or
product can be transcribed from a transcription unit and thus
expressed. Each transcription unit contains the regulatory elements
which are necessary for the transcription and translation of the
gene sequences contained therein. Each transcription unit may
contain the same or different regulatory elements. IRES elements or
introns may be used for the functional linking of the genes within
a transcription unit.
[0121] The expression vector may contain a single transcription
unit for expressing the gene (or genes) of interest, the selectable
marker and optionally the gene which codes for the fluorescent
protein. Alternatively, these genes may also be arranged in two or
more transcription units. Various combinations of the genes within
a transcription unit are possible. In another embodiment of the
present invention more than one expression vector consisting of
one, two or more transcription units may be inserted in a host cell
by cotransfection or in successive transfections in any desired
order. Any combination of regulatory elements and genes on each
vector can be selected provided that adequate expression of the
transcription units is ensured. If necessary, other regulatory
elements, such as TE elements, and genes, e.g. additional genes of
interest or selectable markers, may be positioned on the expression
vectors.
[0122] Also preferred according to the invention are those
expression vectors which contain one or more TE elements and
instead of the gene of interest have only a multiple cloning site
which allows the cloning of the gene of interest via recognition
sequences for restriction endonucleases. Numerous recognition
sequences for all kinds of restriction endonucleases as well as the
associated restriction endonucleases are known from the prior art.
Preferably, sequences are used which consist of at least six
nucleotides as recognition sequence. A list of suitable recognition
sequences can be found for example in Sambrook et al., 1989.
[0123] Also preferred according to the invention are those
expression vectors which instead of the gene of interest have only
a multiple cloning site which allows the cloning of the gene of
interest via recognition sequences for restriction endonucleases
and which moreover have one or more, preferably multiple cloning
sites at different positions of the expression vector, which
additionally makes it possible to clone TE elements via recognition
sequences for restriction endonucleases. Numerous recognition
sequences for all kinds of restriction endonucleases as well as the
associated restriction endonucleases are known from the prior art.
Preferably, sequences are used which consist of at least six
nucleotides as recognition sequence. A list of suitable recognition
sequences can be found for example in Sambrook et al., 1989.
Host Cells:
[0124] For transfection with the expression vector according to the
invention eukaryotic host cells are used, preferably mammalian
cells and more particularly rodent cells such as mouse, rat and
hamster cell lines. The successful transfection of the
corresponding cells with an expression vector according to the
invention results in transformed, genetically modified, recombinant
or transgenic cells, which are also the subject of the present
invention.
[0125] Preferred host cells for the purposes of the invention are
hamster cells such as BHK21, BHK TK.sup.-, CHO, CHO-K1, CHO-DUKX,
CHO-DUKX B1 and CHO-DG44 cells or derivatives/descendants of these
cell lines. Particularly preferred are CHO-DG44, CHO-DUKX, CHO-K1
and BHK21 cells, particularly CHO-DG44 and CHO-DUKX cells. Also
suitable are myeloma cells from the mouse, preferably NS0 and Sp2/0
cells and derivatives/descendants of these cell lines.
[0126] Examples of hamster and mouse cells which can be used
according to the invention are given in Table 1 that follows.
However, derivatives and descendants of these cells, other
mammalian cells including but not restricted to cell lines of
humans, mice, rats, monkeys, rodents, or eukaryotic cells,
including but not restricted to yeast, insect, bird and plant
cells, may also be used as host cells for the production of
biopharmaceutical proteins.
TABLE-US-00001 TABLE 1 Hamster and Mouse Production Cell Lines Cell
line Accession Number NS0 ECASS No. 85110503 Sp2/0-Ag14 ATCC
CRL-1581 BHK21 ATCC CCL-10 BHK TK.sup.- ECACC No. 85011423 HaK ATCC
CCL-15 2254-62.2 (BHK-21-derivative) ATCC CRL-8544 CHO ECACC No.
8505302 CHO-K1 ATCC CCL-61 CHO-DUKX ATCC CRL-9096 (=CHO duk.sup.-
CHO/dhfr.sup.-) CHO-DUKX B1 ATCC CRL-9010 CHO-DG44 Urlaub et al;
Cell 32[2], 405-412, 1983 CHO Pro-5 ATCC CRL-1781 V79 ATCC CCC-93
B14AF28-G3 ATCC CCL-14 CHL ECACC No. 87111906
[0127] The transfection of the eukaryotic host cells with a
polynucleotide or one of the expression vectors according to the
invention is carried out by conventional methods (Sambrook et al.,
1989; Ausubel et al., 1994). Suitable methods of transfection
include for example liposome-mediated transfection, calcium
phosphate coprecipitation, electroporation, polycation- (e.g. DEAE
dextran)-mediated transfection, protoplast fusion, microinjection
and viral infections. According to the invention stable
transfection is preferably carried out in which the constructs are
either integrated into the genome of the host cell or an artificial
chromosome/minichromosome, or are episomally contained in stable
manner in the host cell. The transfection method which gives the
optimum transfection frequency and expression of the heterologous
gene in the host cell in question is preferred. By definition,
every sequence or every gene inserted in a host cell is referred to
as a "heterologous sequence" or "heterologous gene" in relation to
the host cell. This applies even if the sequence to be introduced
or the gene to be introduced is identical to an endogenous sequence
or an endogenous gene of the host cell. For example, a hamster
actin gene introduced into a hamster host cell is by definition a
heterologous gene.
[0128] According to the invention, recombinant mammalian cells,
preferably rodent cells, most preferably hamster cells such as CHO
or BHK cells which have been transfected with one of the expression
vectors according to the invention described herein are
preferred.
[0129] In the recombinant production of heteromeric proteins such
as e.g. monoclonal antibodies (mAb), the transfection of suitable
host cells can theoretically be carried out by two different
methods. mAb's of this kind are composed of a number of subunits,
the heavy and light chains. Genes coding for these subunits may be
accommodated in independent or multicistronic transcription units
on a single plasmid with which the host cell is then transfected.
This is intended to secure the stoichiometric representation of the
genes after integration into the genome of the host cell. However,
in the case of independent transcription units it must hereby be
ensured that the mRNAs which encode the different proteins display
the same stability and transcriptional and translational
efficiency. In the second case, the expression of the genes take
place within a multicistronic transcription unit by means of a
single promoter and only one transcript is formed. By using IRES
elements, a highly efficient internal translation initiation of the
genes is obtained in the second and subsequent cistrons. However,
the expression rates for these cistrons are lower than that of the
first cistron, the translation initiation of which, by means of a
so-called "cap"-dependent pre-initiation complex, is substantially
more efficient than IRES-dependent translation initiation. In order
to achieve a truly equimolar expression of the cistrons, additional
inter-cistronic elements may be introduced, for example, which
ensure uniform expression rates in conjunction with the IRES
elements (WO 94/05785).
[0130] Another possible way of simultaneously producing a number of
heterologous proteins, which is preferred according to the
invention, is cotransfection, in which the genes are separately
integrated in different expression vectors. This has the advantage
that certain proportions of genes and gene products with one
another can be adjusted, thereby balancing out any differences in
the mRNA stability and in the efficiency of transcription and
translation. In addition, the expression vectors are more stable
because of their small size and are easier to handle both during
cloning and during transfection.
[0131] In one particular embodiment of the invention, therefore,
the host cells are additionally transfected, preferably
cotransfected, with one or more vectors having genes which code for
one or more other proteins of interest. The other vector or vectors
used for the cotransfection code, for example, for the other
protein or proteins of interest under the control of the same
promoter, preferably under the control of the same
promoter/enhancer combination or, particularly preferably, under
the control of the same promoter/enhancer/TE element combination or
under the control of the same promoter/enhancer combination with
different TE elements and for at least one selectable marker, e.g.
dihydrofolate reductase.
[0132] In another embodiment of the invention the vectors used for
the transfection may contain one or more TE-elements in any
combination, position and orientation.
[0133] In another particular embodiment of the invention the host
cells are co-transfected with at least two eukaryotic expression
vectors, at least one of the two vectors containing at least one
gene which codes for at least the protein of interest, while the
other vector contains one or more nucleic acids according to the
invention in any combination, position and orientation, and
optionally also codes for at least one gene of interest, and these
nucleic acids according to the invention impart their
transcription- or expression-enhancing activity to the genes of
interest which are located on the other co-transfected vector, by
co-integration with the other vector.
[0134] According to the invention the host cells are preferably
established, adapted and cultivated under serum-free conditions,
optionally in media which are free from animal proteins/peptides.
Examples of commercially obtainable media include Ham's F12 (Sigma,
Deisenhofen, Del.), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's
Medium (DMEM; Sigma), Minimal Essential Medium (MEM; Sigma),
Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO
(Invitrogen, Carlsbad, Calif., USA), CHO-S-SFMII (Invitrogen),
serum-free CHO-Medium (Sigma) and protein-free CHO-Medium (Sigma).
Each of these media may optionally be supplemented with various
compounds, e.g. hormones and/or other growth factors (e.g. insulin,
transferrin, epidermal growth factor, insulin-like growth factor),
salts (e.g. sodium chloride, calcium, magnesium, phosphate),
buffers (e.g. HEPES), nucleosides (e.g. adenosine, thymidine),
glutamine, glucose or other is equivalent nutrients, antibiotics
and/or trace elements. Although serum-free media are preferred
according to the invention, the host cells may also be cultivated
using media which have been mixed with a suitable amount of serum.
In order to select genetically modified cells which express one or
more selectable marker genes, one or more selecting agents are
added to the medium.
[0135] The term "selecting agent" refers to a substance which
affects the growth or survival of host cells with a deficiency for
the selectable marker gene in question. Within the scope of the
present invention, geneticin (G418) is preferably used as the
medium additive for the selection of heterologous host cells which
carry a wild-type or preferably a modified neomycin
phosphotransferase gene. Preferably, G418 concentrations of between
100 and 800 .mu.g/ml of medium are used, most preferably 200 to 400
.mu.g G418/ml of medium. If the host cells are to be transfected
with a number of expression vectors, e.g. if several genes of
interest are to be separately introduced into the host cell, they
generally have different selectable marker genes.
[0136] A selectable marker gene is a gene which allows the specific
selection of cells which contain this gene by the addition of a
corresponding selecting agent to the cultivation medium. As an
illustration, an antibiotic resistance gene may be used as a
positive selectable marker. Only cells which have been transformed
with this gene are able to grow in the presence of the
corresponding antibiotic and are thus selected. Untransformed
cells, on the other hand, are unable to grow or survive under these
selection conditions. There are positive, negative and bifunctional
selectable markers. Positive selectable markers permit the
selection and hence enrichment of transformed cells by conferring
resistance to the selecting agent or by compensating for a
metabolic or catabolic defect in the host cell. By contrast, cells
which have received the gene for the selectable marker can be
selectively eliminated by negative selectable markers. An example
of this is the thymidine kinase gene of the Herpes Simplex virus,
the expression of which in cells with the simultaneous addition of
acyclovir or gancyclovir leads to the elimination thereof. The
selectable markers used in this invention, including the
amplifiable selectable markers, include genetically modified
mutants and variants, fragments, functional equivalents,
derivatives, homologues and fusions with other proteins or
peptides, provided that the selectable marker retains its selective
qualities. Such derivatives display considerable homology in the
amino acid sequence in the regions or domains which are deemed to
be selective. The literature describes a large number of selectable
marker genes including bifunctional(positive/negative) markers (see
for example WO 92/08796 and WO 94/28143). Examples of selectable
markers which are usually used in eukaryotic cells include the
genes for aminoglycoside phosphotransferase (APH), hygromycine
phosphostransferase (HYG), dihydrofolate reductase (DHFR),
thymidine kinase (TK), glutamine synthetase, asparagin synthetase
and genes which confer resistance to neomycin (G418), puromycin,
histidinol D, bleomycin, phleomycin and zeocin.
[0137] The term "modified neomycin-phosphotransferase (NPT)" covers
all the mutants described in WO2004/050884, particularly the mutant
D227G (Asp227Gly), which is characterised by the substitution of
aspartic acid (Asp, D) for glycine (Gly, G) at amino acid position
227 and particularly preferably the mutant F240I (Phe240Ile), which
is characterised by the substitution of phenylalanine (Phe, F) for
isoleucine (Ile, I) at amino acid position 240.
[0138] The present invention therefore includes a method of
preparing and selecting recombinant mammalian cells which comprises
the following steps: (i) transfecting the host cells with genes
which code for at least one protein/product of interest and a
neomycin-phosphotransferase, preferably modified, wherein in order
to enhance the transcription or expression at least the gene (or
genes) of interest is functionally linked to at least one TE
element; (ii) cultivating the cells under conditions that enable
expression of the different genes; and (iii) selecting these
co-integrated genes by cultivating the cells in the presence of a
selecting agent such as e.g. G418. Preferably, the transfected
cells are cultivated in medium in the absence of serum. Preferably
the concentration of G418 is at least 200 .mu.g/mL. However, the
concentration may also be at least 400 .mu.g/mL.
Amplifiable Selectable Marker Gene:
[0139] In addition, the cells according to the invention may
optionally also be subjected to one or more gene amplification
steps in which they are cultivated in the presence of a selecting
agent which leads to amplification of an amplifiable selectable
marker gene.
[0140] The prerequisite is that the host cells are additionally
transfected with a gene which codes for an amplifiable selectable
marker. It is conceivable for the gene which codes for an
amplifiable selectable marker to be present on one of the
expression vectors according to the invention or to be introduced
into the host cell by means of another vector.
[0141] The amplifiable selectable marker gene usually codes for an
enzyme which is needed for the growth of eukaryotic cells under
certain cultivation conditions. For example, the amplifiable
selectable marker gene may code for dihydrofolate reductase (DHFR).
In this case the gene is amplified if a host cell transfected
therewith is cultivated in the presence of the selecting agent
methotrexate (MTX).
[0142] The following Table 2 gives examples of other amplifiable
selectable marker genes and the associated selecting agents which
may be used according to the invention, which are described in an
overview by Kaufman, Methods in Enzymology, 185:537-566 (1990).
TABLE-US-00002 TABLE 2 Amplifiable selectable marker genes
Amplifiable selectable marker gene Accession number Selecting agent
dihydrofolate reductase M19869 (hamster) methotrexate (MTX) E00236
(mouse) metallothionein D10551 (hamster) cadmium M13003 (human)
M11794 (rat) CAD (carbamoylphosphate M23652 (hamster)
N-phosphoacetyl-L-aspartate synthetase:aspartate D78586 (human)
transcarbamylase: dihydroorotase) adenosine-deaminase K02567
(human) Xyl-A- or adenosine, M10319 (mouse) 2'deoxycoformycin AMP
(adenylate)-deaminase D12775 (human) adenine, azaserin, coformycin
J02811 (rat) UMP-synthase J03626 (human) 6-azauridine, pyrazofuran
IMP 5'-dehydrogenase J04209 (hamster) mycophenolic acid J04208
(human) M33934 (mouse) xanthine-guanine- X00221 (E. coli)
mycophenolic acid with phosphoribosyltransferase limiting xanthine
mutant HGPRTase or mutant J00060 (hamster) hypoxanthine,
aminopterine thymidine-kinase M13542, K02581 (human) and thymidine
(HAT) J00423, M68489(mouse) M63983 (rat) M36160 (Herpes virus)
thymidylate-synthetase D00596 (human) 5-fluorodeoxyuridine M13019
(mouse) L12138 (rat) P-glycoprotein 170 (MDR1) AF016535 (human)
several drugs, e.g. J03398 (mouse) adriamycin, vincristin,
colchicine ribonucleotide reductase M124223, K02927 (mouse)
aphidicoline glutamine-synthetase AF150961 (hamster) methionine
sulphoximine U09114, M60803 (mouse) (MSX) M29579 (rat)
asparagine-synthetase M27838 (hamster) .beta.-aspartylhydroxamate,
M27396 (human) albizziin, 5'azacytidine U38940 (mouse) U07202 (rat)
argininosuccinate- synthetase X01630 (human) canavanin M31690
(mouse) M26198 (bovine) ornithine-decarboxylase M34158 (human)
.alpha.-difluoromethylornithine J03733 (mouse) M16982 (rat)
HMG-CoA-reductase L00183, M12705 (hamster) compactin M11058 (human)
N-acetylglucosaminyl- M55621 (human) tunicamycin transferase
threonyl-tRNA-synthetase M63180 (human) borrelidin
Na.sup.+K.sup.+-ATPase J05096 (human) ouabain M14511 (rat)
[0143] According to the invention the amplifiable selectable marker
gene used is preferably a gene which codes for a polypeptide with
the function of DHFR, e.g. for DHFR or a fusion protein from the
fluorescent protein and DHFR. DHFR is necessary for the
biosynthesis of purines. Cells which lack the DHFR genes cannot
grow in purine-deficient medium. The DHFR gene is therefore a
useful selectable marker for selecting and amplifying genes in
cells cultivated in purine-free medium. The selecting medium used
in conjunction with the DHFR gene is methotrexate (MTX).
[0144] Mammalian cells, preferably mouse myeloma and hamster cells,
are preferred host cells for the use of DHFR as an amplifiable
selectable marker. The cell lines CHO-DUKX (ATCC CRL-9096) and
CHO-GD44 (Urlaub et al., 1983) are particularly preferred as they
have no DHFR activity of their own, as a result of mutation. In
order to be able to use the DHFR-induced amplification in other
cell types as well which have their own endogenous DHFR activity,
it is possible to use in the transfection process a mutated DHFR
gene which codes for a protein with reduced sensitivity to
methotrexate (Simonson et al., 1983; Wigler et al., 1980; Haber et
al., 1982).
[0145] The DHFR marker is particularly suitable for the selection
and subsequent amplification when using DHFR-negative basic cells
such as CHO-DG44 or CHO-DUKX, as these cells do not express
endogenous DHFR and therefore do not grow in purine-free medium.
Consequently, the DHFR gene may be used here as a dominant
selectable marker and the transformed cells are selected in
hypoxanthine/thymidine-free medium.
[0146] The present invention therefore includes a method of
preparing and selecting recombinant mammalian cells which comprises
the following steps: (i) transfecting the host cells with genes
which code for at least one protein/product of interest and the
amplifiable selectable marker DHFR, wherein in order to enhance the
transcription or expression at least the gene (or genes) of
interest is functionally linked to at least one TE element; (ii)
cultivating the cells under conditions that enable expression of
the different genes; and (iii) amplifying these co-integrated genes
by cultivating the cells in the presence of a selecting agent which
allows the amplification of at least the amplifiable selectable
marker gene, such as methotrexate. Preferably, the transfected
cells are cultivated in hypoxanthine/thymidine-free medium in the
absence of serum and with the addition of increasing concentrations
of MTX. Preferably, the concentration of MTX in the first
amplification step is at least 100 nM. The concentration of MTX
may, however, also be at least 250 nM and may be increased step by
step to 1 .mu.M. In individual cases concentrations of over 1 .mu.M
may also be used, e.g. 2 .mu.M.
[0147] The present invention also includes a method of preparing
and selecting recombinant mammalian cells which comprises the
following steps: (i) transfecting the host cells with genes which
code for at least one protein/product of interest, a
neomycin-phosphotransferase, preferably modified, and the
amplifiable selectable marker DHFR, wherein in order to enhance the
transcription or expression at least the gene (or genes) of
interest is functionally linked to at least one TE element; (ii)
cultivating the cells under conditions that enable expression of
the different genes; (iii) selecting these co-integrated genes by
cultivating the cells in the presence of a selecting agent such as
e.g. G418, in hypoxanthine/thymidine-free medium; and (iv)
amplifying these co-integrated genes by cultivating the cells in
the presence of a selecting agent which allows the amplification of
at least the amplifiable selectable marker gene, such as
methotrexate. Preferably, the transfected cells are cultivated in
hypoxanthine/thymidine-free medium, supplemented with at least 200
.mu.g/mL G418, preferably 400 .mu.g/mL or even more G418, in the
absence of serum and with the addition of increasing concentrations
of MTX. Preferably, the concentration of MTX in the first
amplification step is at least 100 nM. The concentration of MTX
may, however, also be at least 250 nM and may be increased step by
step to 1 .mu.M. In individual cases concentrations of over 1 .mu.M
may also be used, e.g. 2 .mu.M.
[0148] It is also possible to select transformed cells by
fluorescence-activated cell sorting (FACS). For this, bacterial
.beta.-galactosidase, cell surface markers or fluorescent proteins
may be used (e.g. green fluorescent protein (GFP) and the variants
thereof from Aequorea victoria and Renilla reniformis or other
species; red fluorescent proteins and proteins which fluoresce in
other colours and their variants from non-bioluminescent organisms
such as e.g. Discosoma sp., Anemonia sp., Clavularia sp., Zoanthus
sp., Aequorea coerulescens) for the selection of transformed
cells.
Gene Expression and Selection of High-Producing Host Cells:
[0149] The term gene expression relates to the transcription and/or
translation of a heterologous gene sequence in a host cell. The
expression rate can be generally determined, either on the basis of
the quantity of corresponding mRNA which is present in the host
cell or on the basis of the quantity of gene product produced which
is encoded by the gene of interest. The quantity of mRNA produced
by transcription of a selected nucleotide sequence can be
determined for example by northern blot hybridisation,
ribonuclease-RNA-protection, in situ hybridisation of cellular RNA
or by PCR methods (e.g. quantitative PCR) (Sambrook et al., 1989;
Ausubel et al., 1994). Proteins which are encoded by a selected
nucleotide sequence can also be determined by various methods such
as, for example, ELISA, protein A HPLC, western blot,
radioimmunoassay, immunoprecipitation, detection of the biological
activity of the protein, immune staining of the protein followed by
FACS analysis or fluorescence microscopy, direct detection of a
fluorescent protein by FACS analysis or fluorescence microscopy
(Sambrook et al., 1989; Ausubel et al., 1994). These methods makes
it possible for example to investigate whether the TE element of
SEQ ID No.1 according to the invention, or any part, fragment or
region thereof or the derivatives or combinations thereof, lead to
an increase in the transcription or expression of a gene of
interest.
[0150] By "enhanced expression, transcription or productivity" is
meant an enhancement of the expression or synthesis of a
heterologous sequence introduced into a host cell, for example a
gene coding for a therapeutic protein, compared to a control. There
is enhanced expression, transcription or productivity if a cell
according to the invention is cultivated by a method described here
according to the invention, and if this cell has at least a
doubling of the specific productivity. There is also enhanced
expression, transcription or productivity if the cell according to
the invention has at least a tripling of the specific productivity.
There is particularly enhanced expression, transcription or
productivity if the cell according to the invention has at least a
quadrupling of the specific productivity. There is particularly
enhanced expression, transcription or productivity if the specific
productivity of the cell according to the invention is increased at
least five-fold. There is particularly enhanced expression,
transcription or productivity if the specific productivity of the
cell according to the invention is increased at least six-fold.
There is particularly enhanced expression, transcription or
productivity if the specific productivity of the cell according to
the invention is increased at least seven-fold.
[0151] Enhanced expression, transcription or productivity can be
achieved both by using one of the expression vectors according to
the invention and by using one of the methods according to the
invention.
[0152] The corresponding processes may be combined with a
FACS-assisted selection of recombinant host cells which contain, as
additional selectable marker, one or more fluorescent proteins
(e.g. GFP) or a cell surface marker. Other methods of obtaining
increased expression, and a combination of different methods may
also be used, are based for example on the use of cis-active
elements for manipulating the chromatin structure (e.g. LCR, UCOE,
EASE, isolators, S/MARs, STAR elements), on the use of (artificial)
transcription factors, treatment of the cells with natural or
synthetic agents for up-regulating endogenous or heterologous gene
expression, improving the stability (half-life) of mRNA or the
protein, improving the initiation of mRNA translation, increasing
the gene dose by the use of episomal plasmids (based on the use of
viral sequences as replication origins, e.g. SV40, polyoma,
adenovirus, EBV or BPV), the use of amplification-promoting
sequences (Hemann et al., 1994) or in vitro amplification systems
based on DNA concatemers (Monaco et al., 1996).
[0153] In a further embodiment the present invention thus also
relates to processes for obtaining and selecting recombinant
mammalian cells which express at least one heterologous gene of
interest and are characterised in that (i) recombinant mammalian
cells are transfected with an expression vector according to the
invention and the gene for an amplifiable selectable marker gene;
(ii) the mammalian cells are cultivated under conditions which
allow expression of the gene or genes of interest, the modified
neomycin phosphotransferase gene and the gene which codes for a
fluorescent protein; (iii) the mammalian cells are cultivated in
the presence of at least one selecting agent which acts selectively
on the growth of mammalian cells and gives preference to the growth
of those cells which express the neomycin phosphotransferase gene;
(iv) the mammalian cells which exhibit high expression of the
fluorescent protein are sorted out by flow-cytometric analysis; (v)
the sorted cells are cultivated under conditions under which the
amplifiable selectable marker gene is expressed; and (vi) a
selecting agent is added to the culture medium which results in the
amplification of the amplifiable selectable marker gene.
[0154] Also preferred according to the invention is a process in
which production cells are replicated and used to prepare the
coding gene product of interest. For this, the selected high
producing cells are preferably cultivated in a serum-free culture
medium and preferably in suspension culture under conditions which
allow expression of the gene of interest. The protein/product of
interest is preferably obtained from the cell culture medium as a
secreted gene product. If the protein is expressed without a
secretion signal, however, the gene product may also be isolated
from cell lysates. In order to obtain a pure homogeneous product
which is substantially free from other recombinant proteins and
host cell proteins, conventional purification procedures are
carried out. First of all, cells and cell debris are removed from
the culture medium or lysate. The desired gene product can then be
freed from contaminating soluble proteins, polypeptides and nucleic
acids, e.g. by fractionation on immunoaffinity and ion exchange
columns, ethanol precipitation, reversed phase HPLC or
chromatography on Sephadex, silica or cation exchange resins such
as DEAE. Methods which result in the purification of a heterologous
protein expressed by recombinant host cells are known to the
skilled man and described in the literature, e.g. by Harris et al.,
1995 and Scopes 1988.
Compositions According to the Invention
[0155] The present invention relates to a nucleic acid which
contains TE-13 (SEQ ID No. 15) contains or a fragment of TE-13 (SEQ
ID No. 15) or the complementary nucleotide sequences thereof or a
derivative of TE-13 (SEQ ID No. 15) or a fragment thereof or the
complementary nucleotide sequences thereof, and which on
chromosomal integration leads to an increase in the transcription
or expression of a gene of interest in an expression system.
[0156] The present invention relates to a nucleic acid which
contains TE-13 (SEQ ID No. 15) or a fragment of TE-13 (SEQ ID No.
15) or the complementary nucleotide sequences thereof or a
derivative of TE-13 (SEQ ID No. 15) or a fragment thereof or the
complementary nucleotide sequences thereof, and which on
chromosomal integration leads to an increase in the transcription
or expression of a gene of interest in an expression system, with
the proviso that the fragment comprises at least one sequence
region from the nucleic acid region between 1 bp and 1578 bp (in
relation to SEQ ID No. 01).
[0157] The present invention relates to a nucleic acid which
contains TE-13 (SEQ ID No. 15) or a fragment of TE-13 (SEQ ID No.
15) or the complementary nucleotide sequences thereof or a
derivative of TE-13 (SEQ ID No. 15) or a fragment thereof or the
complementary nucleotide sequences thereof, and which on
chromosomal integration leads to an increase in the transcription
or expression of a gene of interest in an expression system, with
the proviso that the fragment comprises at least one sequence
region of TE-09 (SEQ ID No. 11) or TE-08 (SEQ ID No. 11) or TE-13
(SEQ ID No. 15). By a sequence region is meant a nucleic acid
region of at least 10 bp, 15 bp, 20 bp, 50 bp, 100 bp.
[0158] The enhanced expression of the gene of interest can be
measured for example by measuring the product titre by ELISA.
[0159] In a preferred embodiment the invention relates to a nucleic
acid which contains TE-08 (SEQ ID No. 10) or a fragment of TE-08
(SEQ ID No. 10) or the complementary nucleotide sequences thereof
or a derivative of TE-08 (SEQ ID No. 10) or a fragment thereof or
the complementary nucleotide sequences thereof, and which on
chromosomal integration leads to an increase in the transcription
or expression of a gene of interest in an expression system.
[0160] In a preferred embodiment of the above nucleic acid
according to the invention the proviso is that the fragment must
also include at least one sequence region from the nucleic acid
region between 1 bp and 1578 bp (in relation to SEQ ID No. 01).
[0161] In a particularly preferred embodiment of the above nucleic
acid according to the invention there is the proviso that the
fragment also includes at least one sequence region of TE-09 (SEQ
ID No. 11) or TE-08 (SEQ ID No. 11) or TE-13 (SEQ ID No. 15).
[0162] By a sequence region is meant a nucleic acid region of at
least 10 bp, 15 bp, 20 bp, 50 bp, 100 bp.
[0163] In another embodiment the invention relates to a nucleic
acid which contains SEQ ID No. 1 or a fragment of SEQ ID No. 1 or
the complementary nucleotide sequences thereof or a derivative of
SEQ ID No. 1 or a fragment thereof or the complementary nucleotide
sequences thereof, which on chromosomal integration leads to an
increase in the transcription or expression of a gene of interest
in an expression system.
[0164] In a preferred embodiment of the above-mentioned nucleic
acid according to the invention there is the proviso that the
fragment also comprises at least one sequence region from the
nucleic acid region between 1 bp and 1578 bp (in relation to SEQ ID
No. 01).
[0165] In a particularly preferred embodiment of the
above-mentioned nucleic acid according to the invention there is
the proviso that the fragment also comprises at least one sequence
region of TE-09 (SEQ ID No. 11) or TE-08 (SEQ ID No. 11) or TE-13
(SEQ ID No. 15). By a sequence region is meant a nucleic acid
region of at least 10 bp, 15 bp, 20 bp, 50 bp, 100 bp.
[0166] The present invention also relates to a nucleic acid (=TE
element) for increasing the transcription or expression of a gene
of interest with SEQ ID No. 1 or a fragment or derivative thereof
or the complementary nucleotide sequences thereof, which leads to
an increase in the transcription or expression of a gene of
interest.
[0167] The increase in the expression of the gene of interest can
be measured for example by measuring the product titre by
ELISA.
[0168] The present invention particularly relates to a nucleic acid
according to the invention, which hybridises under stringent
conditions
(a) with the region of nucleic acid sequence TE-13 (SEQ ID No. 15)
or TE-08 (SEQ ID No. 10) or (b) the complementary nucleic acid
sequences thereof or (c) a nucleic acid sequence which has at least
about 70% sequence identity, preferably at least about 80% sequence
identity, preferably at least about 85% sequence identity, most
preferably at least about 90% sequence identity and most preferably
at least about 95% sequence identity with (a) or (b),
[0169] In a special embodiment the nucleic acid according to the
invention has a length of at least 511 bp (=length TE-13, SEQ ID
No. 15) or at least 1015 by (=length TE-08, SEQ ID No. 10).
[0170] In a preferred embodiment the present invention relates to
the 5' fragment of the TE element TE-00 (SEQ ID No. 2). This
corresponds to the part of SEQ ID No. 1 between 1 by and 1578 bp or
the complementary nucleotide sequence thereof.
[0171] The present invention particularly relates to a nucleic acid
or a transcription-enhancing or expression-enhancing nucleic acid
element (TE element), which contains TE-13 (SEQ ID No. 15) or TE-08
(SEQ ID No. 10) or a derivative thereof or the complementary
nucleotide sequences thereof, which on chromosomal integration
leads to an increase in the transcription or expression of a gene
of interest.
[0172] The present invention preferably relates to an isolated
nucleic acid or an isolated nucleic acid molecule or an isolated
nucleic acid sequence or an isolated transcription-enhancing
nucleic acid element or an isolated TE element.
[0173] The present invention particularly relates to an isolated
nucleic acid which contains TE-08 (SEQ ID No. 10) or the
complementary nucleotide sequence thereof and which on chromosomal
integration leads to an increase in the transcription or expression
of a gene of interest in an expression system.
[0174] In one embodiment the nucleic acid or the
transcription-enhancing nucleic acid element or the isolated
nucleic acid contains a derivative of a TE element or of SEQ ID No.
1, which has at least about 70% sequence identity, preferably at
least about 80% sequence identity, most preferably at least about
90% sequence identity and most preferably at least about 95%
sequence identity with the corresponding part of the TE element
sequence or the complementary sequence thereof, particularly with
the sequence region between nucleotide position 1 bp and 1578 bp in
relation to SEQ ID No. 1, corresponding to the sequence region 5'
of the TE-00 sequence, and particularly preferably with TE-13 (SEQ
ID No. 15) or TE-08 (SEQ ID No. 10).
[0175] In a preferred embodiment the nucleic acid or the
transcription-enhancing nucleic acid element or the isolated
nucleic acid contains a derivative of a TE-08 nucleic acid (SEQ ID
No. 10) or preferably of a TE-13 nucleic acid (SEQ ID No. 15),
which has at least about 70% sequence identity, preferably at least
about 80% sequence identity, most preferably at least about 90%
sequence identity and most preferably at least about 95% sequence
identity with the corresponding part of the TE element sequence or
the complementary sequence thereof.
[0176] In another embodiment the invention relates to a nucleic
acid or a transcription-enhancing nucleic acid element or an
isolated nucleic acid or a derivative of a TE element, which
hybridise(s) with the sequence of a TE element or with the
complementary sequence of a TE element, particularly with the
sequence region between nucleotide position 1 bp and 1578 bp in
relation to SEQ ID No. 1, corresponding to sequence region 5' of
the TE-00 sequence (SEQ ID No. 2), or hybridises particularly with
the TE-08 element (SEQ ID No.). Preferably the hybridisation is
carried out under stringent hybridisations and washing
conditions.
[0177] In another preferred embodiment the nucleic acid according
to the invention or the transcription-enhancing element (TE
element) is selected from among: TE-00 (SEQ ID No. 2), TE-01 (SEQ
ID No. 3), TE-02 (SEQ ID No. 4), TE-03 (SEQ ID No. 5), TE-04 (SEQ
ID No. 6), TE-06 (SEQ ID No. 8), TE-07 (SEQ ID No. 9), TE-08 (SEQ
ID No. 10), TE-10 (SEQ ID No. 12), TE-11 (SEQ ID No. 13), TE-12
(SEQ ID No. 14), TE-13 (SEQ ID No. 15), TE-14 (SEQ ID No. 16),
TE-15 (SEQ ID No. 17), TE-16 (SEQ ID No. 18), TE-17 (SEQ ID No.
19), TE-18 (SEQ ID No. 20) and TE-21 (SEQ ID No. 21).
[0178] In another embodiment the nucleic acid or the
transcription-enhancing element (TE element) is characterised in
that the nucleic acid is TE-00 (SEQ ID No. 2), TE-06 (SEQ ID No.
8), TE-10 (SEQ ID No. 12), TE-11 (SEQ ID No. 13) or TE-12 (SEQ ID
No. 14), preferably TE-06 (SEQ ID No. 8).
[0179] In a preferred embodiment the nucleic acid or the
transcription-enhancing element (TE element) is characterised in
that the nucleic acid is TE-01 (SEQ ID No. 3), TE-02 (SEQ ID No.
4), TE-03 (SEQ ID No. 5),TE-07 (SEQ ID No. 9), TE-08 (SEQ ID No.
10).
[0180] In a particularly preferred embodiment the nucleic acid or
the transcription-enhancing element is TE-08 (SEQ ID No. 10).
[0181] In another embodiment the nucleic acid or the
transcription-enhancing element (TE element) is characterised in
that it is a fragment or derivative of TE-01 (SEQ ID No. 3) is,
preferably TE-13 (SEQ ID No. 15), TE-14 (SEQ ID No. 16), TE-15 (SEQ
ID No. 17), TE-16 (SEQ ID No. 18), TE-17 (SEQ ID No. 19), TE-18
(SEQ ID No. 20).
[0182] In a preferred embodiment the nucleic acid according to the
invention is TE-13 (SEQ ID No. 15).
[0183] In another embodiment the nucleic acid according to the
invention or the fragment or the derivative is an isolated nucleic
acid.
[0184] The present invention also relates to a nucleic acid
according to the invention according to one of claims 1 to 12,
characterised in that a nucleic acid containing TE-13 (SEQ ID No.
15) or TE-08 (SEQ ID No. 10) or TE-07 (SEQ ID No. 9) or TE-06 (SEQ
ID No.8) or a fragment of these sequences or the complementary
nucleotide sequences thereof or a derivative of these sequences or
a derivative of fragments of these sequences, preferably TE-13 (SEQ
ID No. 15) or TE-08 (SEQ ID No. 10) or a fragment of these
sequences or a derivative of these sequences or a derivative of
fragments of these sequences or the complementary nucleotide
sequences thereof, is linked to a heterologous sequence.
[0185] The nucleic acid linked to a heterologous gene sequence may
in a preferred embodiment be an expression vector, for example
plasmids, bacteriophages, phagemids, cosmids, viral vectors or
particularly a targeting vector. The nucleic acid linked to a
heterologous gene sequence may however also be any other synthetic
nucleic acid molecule such as e.g. synthetic, artificial or
mini-chromosomes.
[0186] The present invention further relates to a eukaryotic
expression vector, characterised in that this expression vector
contains one or more nucleic acids according to the invention or
one or more transcription-enhancing elements (TE element) according
to the invention.
[0187] In a special embodiment the eukaryotic expression vector is
characterised in that it contains a promoter and/or a heterologous
gene of interest and/or a selectable marker and/or optionally an
enhancer.
[0188] The present invention further relates to a eukaryotic
expression vector characterised in that this expression vector
contains one or more nucleic acids according to the invention or
one or more transcription-enhancing elements (TE element) and a
promoter and/or a heterologous gene of interest and/or a selectable
marker and/or optionally an enhancer according to the
invention.
[0189] In another embodiment the eukaryotic expression vector is
characterised in that it is a targeting vector for the deliberate
integration of the gene of interest.
[0190] In a preferred embodiment the eukaryotic expression vector
is characterised in that the promoter is a heterologous promoter
such as the early promoter of human cytomegaly virus
(CMV-promoter), SV40 early promoter, adenovirus major late
promoter, mouse metallothionein-I promoter, the long terminal
repeat region of Rous Sarcoma Virus, actin-, immunoglobulin or
heat-shock-promotor(s), preferably CMV-promoter.
[0191] In another preferred embodiment the eukaryotic expression
vector is characterised in that the promoter is a heterologous
promoter, preferably ubiquitin/S27a-promoter, most preferably
hamster ubiquitin/S27a-promoter.
[0192] In a special embodiment the eukaryotic expression vector is
characterised in that it contains a combination of several
identical or different nucleic acids or TE-elements according to
the invention in any orientation to one another, wherein one or
more nucleic acids or TE-elements are positioned in front of (i.e.
5' of) and/or one or more nucleic acids or TE-elements are
positioned (i.e. 3' of) the gene of interest.
[0193] In a preferred embodiment the eukaryotic expression vector
is characterised in that the combined nucleic acids or TE elements
are TE-06 (SEQ ID No. 8) and particularly preferably TE-08 (SEQ ID
No. 10).
[0194] Preferred combinations of nucleic acids or TE elements are a
TE-06 element (SEQ ID No. 8) in front of (i.e. 5' of) and a TE-06
element (SEQ ID No. 8) behind (i.e. 3' of) the gene of interest and
three TE-06-elements (SEQ ID No. 8) behind (i.e. 3' of) the gene of
interest (cf also FIG. 13).
[0195] In a particularly preferred embodiment the eukaryotic
expression vector is characterised in that one or more
TE-08-nucleic acid(s) or element(s) (SEQ ID No. 10) are positioned
in front of (i.e. 5' of) and one or more behind (i.e. 3' of) the
gene of interest, preferably one TE-08 element (SEQ ID No. 10) in
front and one behind (cf also FIG. 13).
[0196] Other preferred combinations of TE-nucleic acids or elements
are 2 TE-08 nucleic acids/elements (SEQ ID No. 10) before and/or
after the gene of interest.
[0197] In another embodiment the eukaryotic expression vector is
characterised in that a plurality of TE-06-nucleic acids or
elements (SEQ ID No. 8) are positioned behind (3' of) the gene of
interest, preferably 3 (cf FIG. 13).
[0198] In a preferred embodiment the eukaryotic expression vector
is characterised in that a combination of one or more TE-08-nucleic
acid(s) or element(s) (SEQ ID No. 10) with one or more TE-06
nucleic acids or element(s) (SEQ ID No. 8) are positioned in front
of (i.e. 5' of) and/or behind (i.e. 3' of) the gene of interest;
the preferred combination is a combination of a TE-08 nucleic acid
or element (SEQ ID No. 10) followed by a TE-06-nucleic acid or
element (SEQ ID No. 8) in front of (i.e. 5' of) the gene of
interest. (Cf also FIG. 13).
[0199] In a preferred embodiment the eukaryotic expression vector
is characterised in that it additionally contains an integrase.
[0200] In another preferred embodiment the host cells are
co-transfected with at least two eukaryotic expression vectors,
while at least one of the two vectors contains at least one gene
which codes for at least one protein of interest and the other
vector contains one or more nucleic acid according to the
inventions in any combination, position and orientation, and
optionally also codes for at least one gene of interest, and these
nucleic acids according to the invention impart their
transcription- or expression-enhancing effect to the genes of
interest which are located on the other co-transfected vector, by
co-integration with the other vector.
[0201] In a special embodiment the eukaryotic expression vector is
characterised in that the selectable marker is DHFR or Neo, for
example Neo F240I.
[0202] The invention further relates to a method of preparing a
eukaryotic expression vector, characterised by the integration of a
nucleic acid according to the invention in an expression
vector.
[0203] The invention further relates to a eukaryotic host cell
characterised in that it contains a eukaryotic expression vector
according to the invention.
[0204] In a special embodiment the eukaryotic host cell is
characterised in that it is a high producer, i.e. it has a higher
specific productivity than a comparable eukaryotic host cell
without a TE element or nucleic acid according to the invention,
this host cell having an expression level which is increased
two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold or
ten-fold or one which is increased more than two-fold, more than
three-fold, more than four-fold, more than five-fold, more than
seven-fold or more than ten-fold, preferably up to five-fold or
more than three-fold.
[0205] In a particularly preferred embodiment the eukaryotic host
cell is characterised in that the expression vector is stably
integrated in the genome.
[0206] In another embodiment the eukaryotic host cell is a hamster
or mouse cell such as for example a CHO, NS0, Sp2/0-Ag14, BHK21,
BHK TK.sup.-, HaK, 2254-62.2 (BHK-21-derivative), CHO-K1,
CHO-DUKX(=CHO duk.sup.-, CHO/dhfr.sup.-), CHO-DUKX B1, CHO-DG44,
CHO Pro-5, V79, B14AF28-G3, CHL cell, preferably a CHO cell and
particularly preferably a CHO-DG44 cell.
[0207] In another embodiment the eukaryotic host cell is a
mammalian cell, including but not restricted to human, mouse, rat,
monkey or rodent cell lines.
[0208] In another embodiment the host cell is a eukaryotic cell
including but not restricted to yeast, insect, bird and plant
cells.
[0209] In another special embodiment the eukaryotic host cell is
characterised in that it additionally contains an anti-apoptosis
gene such as BCL-xL, BCL-2, BCL-w, BFL-1, A1, MCL-1, BOO, BRAG-1,
NR-13, CDN-1, CDN-2, CDN-3, BHRF-1, LMW5-HL or CED-9, preferably
Bcl-xL or BCL-2, most preferably BCL-xL.
[0210] The present invention further relates to a method of
developing a high-producing stably transfected eukaryotic host cell
line, characterised by the following steps:
(a) integrating at least one nucleic acid according to the
invention or one TE element according to the invention in a
eukaryotic expression vector containing a gene of interest, (b)
transfecting a eukaryotic host cell with this expression vector,
(c) selecting a highly-productive transfected host cell.
[0211] The present invention further relates to a method of
developing a high-producing stably transfected eukaryotic host cell
line, characterised by the following steps:
(a) integrating a gene (genes) of interest in a eukaryotic
expression vector containing at least one nucleic acid according to
the invention or a TE element according to the invention (b)
transfecting a eukaryotic host cell with this expression vector,
(c) selecting a highly-productive transfected host cell.
[0212] In a special embodiment the method is characterised by at
least one additional amplification step.
[0213] The present invention also relates to a method of preparing
and selecting recombinant mammalian cells, characterised by the
following steps:
(a) transfecting the host cells with genes that codes at least for
a protein/product of interest, a neomycin-phosphotransferase,
preferably modified, and the amplifiable selectable marker DHFR,
wherein in order to enhance the transcription or expression at
least the gene (or genes) of interest is or are functionally linked
to at least one nucleic acid according to the invention, (b)
cultivating the cells under conditions which enable expression of
the different genes, (c) selecting these co-integrated genes by
cultivating the cells in the presence of a selecting agent, such as
e.g. G418, in a hypoxanthine/thymidine-free medium and (d)
amplifying these co-integrated genes by cultivating the cells in
the presence of a selecting agent which allows the amplification of
at least the amplifiable selectable marker gene, such as e.g.
methotrexate.
[0214] In a particular embodiment this method is characterised in
that the transfected cells are cultivated in
hypoxanthine/thymidine-free medium, supplemented with at least 200
.mu.g/mL G418, preferably 400 .mu.g/mL or more G418, in the absence
of serum and with the addition of increasing concentrations of
MTX.
[0215] In another particular embodiment this method is
characterised in that the concentration of MTX in the first
amplification step is at least 100 nM or at least 250 nM and is
increased stepwise to 1 .mu.M or above. In individual cases, the
MTX concentration may be 2 .mu.M.
[0216] In another special embodiment the method is characterised by
an additional cloning step.
[0217] In another embodiment of the method according to the
invention the host cell is a rodent/hamster cell such as for
example a CHO, NS0, Sp2/0-Ag14, BHK21, BHK TK.sup.-, HaK, 2254-62.2
(BHK-21-derivative), CHO-K1, CHO-DUKX(=CHO duk.sup.-,
CHO/dhfr.sup.-), CHO-DUKX B1, CHO-DG44, CHO Pro-5, V79, B14AF28-G3,
CHL cell, preferably a CHO cell and particularly preferably a
CHO-DG44 cell.
[0218] In a preferred method according to the invention the
expression vector contains a selectable marker such as DHFR or NPT,
for example NPT F240I or NPT D227G.
[0219] In a particularly preferred embodiment of the method
according to the invention the proportion of high producers is
increased up to two-fold, three-fold, four-fold, five-fold,
six-fold, seven-fold or ten-fold or more than two-fold, more than
three-fold, more than four-fold, more than five-fold, more than
seven-fold or more than ten-fold, preferably up to five-fold or
more than three-fold.
[0220] The present invention further relates to a method of
preparing a biopharmaceutical product, characterised by the
following steps:
(a) integrating at least one nucleic acid according to the
invention or one TE element according to the invention in a
eukaryotic expression vector containing a gene of interest, (b)
transfecting a eukaryotic host cell with this expression vector,
(c) selecting a highly-productive transfected host cell and (d)
cultivating the highly-productive transfected host cell obtained
under conditions which allow expression of the gene(s) of
interest.
[0221] The present invention further relates to a method of
developing a high-producing stably transfected eukaryotic host cell
line, characterised by the following steps:
(a) integrating a gene (genes) of interest in a eukaryotic
expression vector containing at least one nucleic acid according to
the invention or one TE element according to the invention (b)
transfecting a eukaryotic host cell with this expression vector,
(c) selecting a highly-productive transfected host cell and (d)
cultivating the highly-productive transfected host cell obtained
under conditions which allow expression of the gene(s) of
interest.
[0222] In a special embodiment the method according to the
invention is characterised by at least one additional amplification
step.
[0223] In a special embodiment the method according to the
invention is characterised by the following additional step:
(e) harvesting and purifying the protein of interest.
[0224] The present invention further relates to the use of a
nucleic acid according to the invention or of a
transcription-enhancing element (TE element) according to the
invention in a eukaryotic expression vector, for increasing the
transcription or expression of a gene of interest in an expression
system in a eukaryotic host cell or for preparing a
biopharmaceutical product.
[0225] The present invention also relates to the use of a nucleic
acid according to the invention or of a transcription-enhancing
element (TE element) according to the invention for producing
transgenic animals or plants.
[0226] The present invention further relates to the use of a
nucleic acid according to the invention or of a
transcription-enhancing element (TE element) according to the
invention in gene therapy.
[0227] The present invention particularly relates to the use of a
nucleic acid according to the invention or of a
transcription-enhancing element (TE element) according to the
invention as a medicament or in a pharmaceutical composition.
[0228] The present invention further relates to a kit consisting of
a nucleic acid according to the invention or (a) TE element(s)
according to the invention, optionally expression vector(s),
optionally host cell(s) and optionally transfection reagent(s).
[0229] In a preferred embodiment the present invention relates to a
nucleic acid, particularly an isolated nucleic acid, more precisely
a transcription-enhancing or expression-enhancing nucleic acid
element (TE element) with SEQ ID No. 1 or a fragment or a
derivative thereof, which on chromosomal integration leads to an
increase in the transcription or expression of a gene of interest,
with the exclusion of the TE element TE-00 (SEQ ID No. 2).
[0230] In another embodiment the present invention relates to a
nucleic acid according to the invention with the exclusion of the
TE elements TE-00 (SEQ ID No. 2), TE-04 (SEQ ID No. 6) and TE-06
(SEQ ID No. 8).
[0231] In another embodiment the present invention relates to a
nucleic acid according to the invention with the exclusion of the
TE elements TE-00 (SEQ ID No. 2), TE-04 (SEQ ID No. 6), TE-05 (SEQ
ID No. 7) and TE-06 (SEQ ID No. 8).
[0232] The increase in the expression of the gene of interest can
be measured for example by measuring the product titre using
ELISA.
[0233] Another embodiment of the present invention relates to a TE
element, fragment or derivative according to the invention, which
is over 160 by long, preferably over 170 by long. In a special
embodiment the TE element fragment is between 160 bp and 1.2 kb or
between 170 by and 1 kb, preferably over 200 by and between 200 by
and 1 kb long.
[0234] In a preferred embodiment the TE element fragment is in the
part of SEQ ID No. 1 between 1 by and 1578 bp (this corresponds to
a fragment 5' of the element TE-00 (SEQ ID No. 2) and is over 113
by long or over 132 by and preferably over 160 by or over 170 by
long. In a special embodiment the TE element fragment is between
113 by and 1.2 kb or between 132 by and 1.2 kb or between 160 by
and 1.2 kb, preferably over 200 by and between 200 by and 1 kb
long.
[0235] In another embodiment the TE element fragment is present
without any adjacent sequences. By this is meant that the fragment
is not part of a larger sequence or a sequence region, for example
no other sequences are attached in front of (5') or behind (3')
it.
[0236] In another special embodiment the present invention relates
to a nucleic acid according to the invention which does not contain
any CpG islands.
[0237] It is apparent from the following experiments that when
eukaryotic host cells with and without TE element are compared, an
up to seven-fold increase in the relative change in the specific
productivity of the gene of interest can be shown.
[0238] The collection of data on the product titre and specific
productivity showed that on average almost all the cell pools with
TE elements which were fragments or derivatives of SEQ ID No.1
expressed more genes of interest than cell pools without a TE
element. The TE-elements 01 (SEQ ID No. 3), 02 (SEQ ID No. 4) and
08 (SEQ ID No. 10) yielded the highest productivity in two
independent transfection series in which a different selectable
marker (NPT or DHFR) was used in each case. They are capable of
increasing the productivity by a factor of 4-7. Certainly, the TE
elements 01 (SEQ ID No. 3) and 02 (SEQ ID No. 4) at 3 kb and 2.5 kb
are very large for additionally attaching to an expression vector.
Of more interest, on the other hand, is the TE element 08 (SEQ ID
No. 10), which is only 1 kb in size, which is capable of increasing
expression by a factor of 5-6. The TE element 06 (SEQ ID No. 8) in
two transfection series yielded a tripling of the specific
productivity with a size of only 381 bp. It is highly advantageous
to leave the expression vectors as small as possible, as smaller
vectors are generally more stable and are easier to handle both
during cloning and during transfection. Therefore, the TE-elements
06 (SEQ ID No. 8) and 08 (SEQ ID No. 10) are particularly
interesting for use as transcription-promoting elements.
[0239] The following Examples also show that the cell pools which
contained the TE element 03 (SEQ ID No. 5), 04 (SEQ ID No. 6) or 07
(SEQ ID No. 9) showed an approximately 3- to 3.5-fold expression of
the gene of interest and cell pools with the TE elements 10 (SEQ ID
No. 12), 11 (SEQ ID No. 13) or 12 (SEQ ID No. 14) showed an
approximately doubled expression of the gene of interest
[0240] The TE element 08 (SEQ ID No. 10) in conjunction with NPT
F240I as selectable marker, at factor 5, demonstrated the greatest
increase in the specific productivity of the gene of interest
compared with the control pools with no TE element. In conjunction
with DHFR as selectable marker the TE element 08 (SEQ ID No. 10)
shows an even better increase of about factor 6.0.
[0241] Furthermore, TE element 02 (SEQ ID No. 4) in the test series
with DHFR as selectable marker shows an increase in the
productivity of the gene of interest by a factor 6.8.
[0242] The Examples that follow also show that pools with the TE
elements 05 (SEQ ID No. 7) and 09 (SEQ ID No. 11) in one test
series exhibited no increase in the expression of the gene of
interest and in one test series even showed a lower expression of
the gene of interest than the control pools. These two elements and
possibly partial fragments in these sequence regions can thus have
a repressive effect under certain circumstances, although this is
not necessarily the case.
[0243] Moreover, in the Examples that follow, the relative changes
in the specific productivity for the different TE-elements tested
are achieved largely independently of the vector system, i.e.
independently of the selectable marker used or independently of the
particular gene of interest.
[0244] The Examples that follow also show that the change in the
expression of the marker gene correlates with the changes in the
expression of the gene of interest.
[0245] It is also apparent from the following Examples that none of
the TE elements tested has an enhancing effect. It is thus clear
that the TE-elements only cause an increase in the expression of
the gene of interest at a chromosomal level.
[0246] The Examples that follow also show that the combination or
concatenation of a plurality of identical or different short
TE-elements such as e.g. TE element 06 and 08 can lead to an
additional expression-enhancing effect.
[0247] The following Examples are provided to illustrate the
present invention, and should not be construed as limiting
thereof.
[0248] All references cited herein are incorporated by reference in
the application in their entireties.
EXAMPLES
Abbreviations
[0249] AP: alkaline phosphatase [0250] Asp (=D): aspartic acid
[0251] bp: base pair [0252] CHO: Chinese Hamster Ovary [0253] DHFR:
dihydrofolate-reductase [0254] ELISA: enzyme-linked immunosorbant
assay [0255] FACS: fluorescence-activated cell sorter [0256] GFP:
green fluorescent protein [0257] Gly (=G): glycine [0258] HT:
hypoxanthine/thymidine [0259] IgG: Immunoglobulin G [0260] Ile
(=I): isoleucine [0261] IRES: internal ribosomal entry site [0262]
kb: kilobase [0263] mAb: monoclonal antibody [0264] MCP-1: monocyte
chemoattractant protein-1 [0265] MTX: methotrexate [0266] NPT:
neomycin-phosphotransferase [0267] PCR: polymerase chain reaction
[0268] Phe (=F): phenylalanine [0269] SEAP: secreted alkaline
phosphatase [0270] Ub: ubiquitin [0271] UTR: untranslated
region
Methods
Cell Culture and Transfection
[0272] The cells CHO-DG44/dhfr.sup.-/- (Urlaub et al., 1983) were
permanently cultivated as suspension cells in serum-free
CHO-S-SFMII medium supplemented with hypoxanthine and thymidine
(HT) (Invitrogen GmbH, Karlsruhe, Del.) in cell culture flasks at
37.degree. C. in a damp atmosphere and 5% CO.sub.2. The cell counts
and viability were determined with a Coulter Counter Z2 (Beckmann
Coulter) or with a Cedex (Innovatis) and the cells were then seeded
in a concentration of 1.times.3.times.10.sup.5/mL and run every 2-3
days.
[0273] Lipofectamine Plus Reagent (Invitrogen GmbH) was used for
the transfection of CHO-DG44. For each transfection mixture a total
of 1.0-1.3 .mu.g of plasmid-DNA, 4 .mu.L of lipofectamine and 6
.mu.L of Plus reagent were mixed together according to the
manufacturer's instructions and added in a volume of 200 .mu.L to
6.times.10.sup.5 cells in 0.8 mL of HT-supplemented CHO-S-SFMII
medium. After three hours' incubation at 37.degree. C. in a cell
incubator 2 mL of HT-supplemented CHO-S-SFMII medium was added.
After a cultivation time of 48 hours, the transfection mixtures
were either harvested (transient transfection) or subjected to
selection. For the NPT-based selection the cells were transferred 2
days after transfection into HT-supplemented CHO-S-SFMII medium
with 400 .mu.g/mL of G418 (Invitrogen). For the DHFR-based
selection, the cells were transferred 2 days after transfection
into HT-free CHO-S-SFMII medium. In DHFR- and NPT-based selection
in the event of co-transfection, in which one expression vector
contained a DHFR and the other expression vector contained an NPT
selectable marker, the cells were transferred 2 days after
transfection into CHO-S-SFMII medium without the addition of
hypoxanthine and thymidine and also G418 (Invitrogen) was added to
the medium in a concentration of 400 .mu.g/mL.
[0274] A DHFR-based gene amplification of the integrated
heterologous genes can be obtained by the addition of the selecting
agent MTX (Sigma, Deisenhofen, Del.) in a concentration of 5-2000
nM to an HT-free CHO-S-SFMII medium.
Expression Vectors
[0275] To analyse the expression, eukaryotic expression vectors
were used which are based on the pAD-CMV vector (Werner et al.,
1998) and mediate the expression of a heterologous gene by the
combination of CMV enhancer/hamster ubiquitin/S27a promoter (WO
97/15664) or CMV enhancer/CMV promoter. While the base vector pBID
contains the dhfr-minigene which acts as an amplifiable selectable
marker (cf e.g. EP-0-393-438), in the vector pBING the
dhfr-minigene has been replaced by a modified NPT gene. This is the
NPT variant D227G (Asp227Gly). The cloning of pBING with the NPT
variant D227G and the IRES-GFP gene region was carried out as
described in (WO2004/050884). The base plasmid pTE4 contains the
NPT variant F240I (Phe240Ile) as selectable marker and is a
derivative of the plasmid pBING. Apart from the replacement of the
NPT variant D227G by the NPT variant F240I the GFP was also
replaced by the red fluorescent protein DsRed2 from the vector
pDsRed2 (Clontech, Palo Alto, Calif., USA). The base plasmid pTE5
contains DHFR as selectable marker and is a derivative of the
vector pBIDG (WO2004/050884), in which again the GFP has been
replaced by the red fluorescent protein DsRed2 from the vector
pDsRed2 (Clontech, Palo Alto, Calif., USA).
[0276] In order to express a monoclonal humanised IgG1 antibody the
heavy chain was cloned as a 1.4 kb SalI/SpeI fragment into the
plasmid pBID digested with XbaI and SalI, to obtain the plasmid
pBID-HC (FIG. 1A). The light chain on the other hand was cloned as
a 0.7 kb BamHI/HindIII fragment into the cutting sites of the
plasmid pBING, producing the plasmid pBING-LC (FIG. 1A).
[0277] Human MCP-1 cDNA (Yoshimura et al., 1989) was cloned into
the corresponding cutting sites of the vector pTE4 or pTE5 as a 0.3
kb HindIII/EcoRI fragment, resulting in the plasmids pTE4/MCP-1 and
pTE5/MCP-1 (FIG. 1B and FIG. 2 respectively).
FACS (Fluorescence-Activated Cell Sorter)
[0278] The flow-cytometric analyses were carried out with a BD
FACScalibur (BD Bioscience). The FACS is fitted with a helium-argon
laser with an excitation wavelength of 488 nm.
[0279] The fluorescence intensity is absorbed at a wavelength
suited to the particular fluorescence protein and processed by
means of the attached software Cell Quest Pro.
Elisa (Enzyme-Linked Immunosorbant Assay)
[0280] The MCP-1 titres in supernatants of stably or transiently
transfected CHO-DG44 cells were quantified by ELISA using the
OptEIA Human MCP-1 Set kit in accordance with the manufacturer's
instructions (BD Biosciences Pharmingen, Heidelberg, Del.).
[0281] The IgG1 mAb in the supernatants from stably transfected
CHO-DG44 cells was quantified by ELISA according to standard
procedures (Current Protocols in Molecular Biology, Ausubel et al.,
1994, updated), using on the one hand a goat anti human IgG Fc
fragment (Dianova, Hamburg, Del.) and on the other hand an
AP-conjugated goat anti human kappa light chain antibody (Sigma).
Purified IgG1 antibody was used as the standard.
[0282] Productivities (pg/cell/day) were calculated by the formula
pg/((Ct-Co) t/In (Ct-Co)), is where Co and Ct are the cell count on
seeding and harvest, respectively, and t is the cultivation
time.
SEAP Assay
[0283] The SEAP titre in culture supernatants from transiently
transfected CHO-DG44 cells was quantified using the SEAP reporter
gene assays in accordance with the manufacturer's instructions
(Roche Diagnostics GmbH, Mannheim, Del.).
Example 1
Isolation and cloning of the TE element TE-A
[0284] Starting from the sequence from the hamster genome described
in WO97/15664, which comprises, in addition to the coding region
for the ubiquitin/S27a gene, adjacent 5'UTR regions including the
Ub/S27a-promoter, hitherto unknown sequence regions were isolated
further upstream. For this, adapter-ligated genomic CHO-DG44 DNA
was used as the matrix for "nested PCRs". The first PCR was carried
out with a combination of primers with complementarity to the
adapter or to a hamster sequence in the 5' region of the sequence
listed in WO97/15664 under SEQ ID No. 5 (primer Ub20:
5'-CTCCACACATTTACACATGGACAC-3' (SEQ ID No. 39)); corresponds to
nucleotides 62 to 85 (complementary sequence) of SEQ ID No. 5 from
WO 97/15664). Then a second PCR was carried out with a second
primer combination, consisting of an inner adapter primer and an
inner ("nested") hamster-specific primer (primer Ub21:
5'-GGGTTTCTCTGTGTAATAGCCATG-3'(SEQ ID No. 40); corresponds to
nucleotides 16 to 39 (complementary sequence) of SEQ ID No.5 from
WO97/15664). The resulting overlapping DNA fragments which started
at the hamster-specific primer end with a known sequence and then
merged into new unknown sequence regions located upstream were
subcloned into pCR2.1 TOPO vectors (Invitrogen) and analysed by
sequencing. In all 348 by of a new, hitherto unknown sequence were
obtained upstream of the hamster Ub/S27a gene.
[0285] On the basis of this new sequence information, another
further upstream DNA region was isolated using the "nested PCR"
described above. This time, the first PCR was carried out with an
adapter primer and the primer Ub33 (5'-ATCTCACTGTGTCTACCAACTTAG-3'
(SEQ ID No. 41); situated in the 5' region of the newly isolated
384 by sequence; corresponds to nucleotide 1268-1291 (complementary
sequence) of SEQ ID No. 1) and the second PCR with an inner adapter
primer and the hamster-specific primer Ub32a located further
inwards (5'-TCTGCACCACCACTACCTGACT-3' (SEQ ID No. 42); located
upstream from the primer Ub33 within the newly isolated 384 by
sequence; corresponds to nucleotide 1243-1264 (complementary
sequence) of SEQ ID No. 1). The amplified material obtained was
subcloned into the pCR2.1 TOPO vector (Invitrogen) and sequenced.
It contained another 1239 by of a new, hitherto unknown sequence
upstream from the hamster Ub/S27a-gene.
[0286] The sequence information from the overlapping PCR fragments
was used to amplify a cohesive sequence region from the genomic DNA
of CHO-DG44 by PCR, which comprised all the partial fragments
hitherto isolated and extended 383 by into the 5' sequence region
of SEQ ID No. 5 from WO 97/15664, using the primers
Ub34 (5'-CTAAGAGTACTTGCCATGAGAGCCTGAA-3' (SEQ ID No. 43); located
at the outermost 5' end of the newly isolated 1239 by sequence;
only partially present in SEQ ID No.1 (nucleotides 1 to 14)) and
Ub35 (5'-CATTGATACACCACCAAAGAACTTG-3' (SEQ ID No. 44); corresponds
to nucleotides 1941 to 1965 (complementary sequence) of SEQ ID No.
1).
[0287] The resulting 2 kb DNA fragment was ligated with the 5' UTR
region of sequence ID No. 5 described in WO 97/15664 via the
endogenous EcoRI cutting site (position 353-358), although this
cutting site was eliminated by a filling reaction with Klenow DNA
polymerase. A second endogenous EcoRI cutting site in the newly
isolated genome region was eliminated in the same way, resulting in
the nucleotides 326 to 329 in SEQ ID No. 1 which are additional to
the original genome sequence. In all, in this way, 8 additional
nucleotides were inserted into SEQ ID No. 1 compared with the
endogenous hamster sequence. The resulting 3788 by DNA fragment
from a sequence region located upstream from the hamster Ub/S27a
gene was designated TE element A with the sequence ID No.1 and
subcloned into the vector pBluescript SKM (Stratagene, La Jolla,
Calif.).
Example 2
Generation of Diverse TE Expression Vectors
[0288] Starting from the 3.8 kb TE-element TE-A (FIG. 3, Sequence
ID No. 1) from the CHO genome various fragments were produced by
PCR which had deletions at either the 5'- or 3'-end, compared with
the TE element TE-A (FIG. 4 and FIG. 5). To synthesise these
fragments combinations of direct and one reverse primer were used
(FIG. 5 and FIG. 6). For cloning purposes a BamHI cutting site was
attached at the 5'-end of the fragment and a BsrGI cutting site was
attached at the 3'-end of the fragment by the primers. In this way
12 TE-elements of different lengths designated TE-01 to TE-12 were
generated (FIGS. 4 and 5). After digestion with BsrGI and BamHI
these were cloned in direct orientation into the adapter region of
the base plasmids pTE4/MCP-1 (FIG. 1B) or pTE5/MCP-1 (FIG. 2). The
fragment TE-00 (SEQ ID No. 2) was isolated from a subclone of TE-A
by SacII-restriction enzyme digestion and cloned into the base
vectors pBING-LC (FIG. 1A) or pBID-HC (FIG. 1A) in both direct and
reverse orientation via the SpeI cutting site located 5' of the
promoter/enhancer element.
Sequences of the TE-Elements
TABLE-US-00003 [0289] TE-A (SEQUENCE ID NO. 1)
ccatgagagcctgaagacctgagttgatacccagaacccagatcaagatggaggagagaaccagccccactaag-
ctgtcccctg
acccccataaatgcctccctgtccagttatgccacacaatgataggtgaatacagaaaaacacccttcctttag-
acactaagcggatt
cctatacgcataccagttaagtgatagttataggatcaactcagcactttaaaaagtttatattttgcaatgct-
ggggactaaattag
ggttgtgcacatgctaagtaagcactctacttttgtatcacattttaataattgtaagaattaattcgtgaaat-
agtagctgagacaatag
atttgtttctttcatgtgggaactgctgtgtgtgcttcttgctgatgcaaacaaggtcaaatactttattcccc-
agtgtctgcctagccctgt
aacacttctctattatacaatgaccacaaataattaggtgagtgggttttgtttcattttaaattgttgctatt-
ttagagacaggatttcttgc
aaacctggttggtcttaaactccgtatgtagctgagaatgaccttgaaaaccttcctgtcccacccctcaaatt-
ccagaattatagaca
cccaccacatggcttaataagtaaacaacaacaataaaagcatgacttctgggtctggagggagggcttgccag-
ttaagagcaatg
gatactttcccatagaacctgggtttgactcccagcactaacctacatggtgatagtgatgcagcagacataca-
tgagggcaacaca
cacatgggcacatacacacgcacccgcccaccatggcttttcccccatcacttagacagccatatttaaacgta-
gtggagccaggc
tggggtggtggcccacacctttaatcccagcactccagaaggcagaggtaggcggatctctgtgggtttgagac-
cagcctggtcta
caagagctagttccaggacagcctccaaagccatagagaaaccctatctcaaaaaactgaaacaacaacaacaa-
caaaacaaaa
taaaaaaacaacaaaagaatcttagtggttcagtggttccacacacaggaaagtagaaagggccttgatgggaa-
ggttttcagagg
gaggagtatggatgagacaggatgatagtgaaaagaactcaaattaattaaatatttgaaactatctaagaata-
aaagctaaaatattt
aaaattacagtcaggtagtggtggtgcagagggctaagttggtagacacagtgagatccaggccagccagggct-
acctagtgag
accttgttcaaataactaataaaatatacaaaataaaggagacaccacaataattttgaaatgtaaaagactaa-
atttaccttttatattg
atgagttggataaaaaaatcaatttaccagagaacataaagtagtcccatcaaagacaaaagcaatatatgatt-
aaactctaatttaaa
agtttgttagagcctggcaacgtggcacatacctttaatcccagcaccagggagacagaggccatcctggtcta-
aaaagtgatctc
caggacagccatggctattacacagagaaaccctgtctggaaaaacaaaaaattagtgtccatgtgtaaatgtg-
tggagtatgcttgt
catgccacatacagaggtagagggcagtttatgggagtcagttcctattcttcctttatgggggacctggggac-
tgaactcaggtcat
caggcttggcagaaagtgcattagctcacggagccttatcattggcgaaagctctctcaagtagaaaatcaatg-
tgtttgctcatagt
gcaatcattatgtttcgagaggggaagggtacaatcgttggggcatgtgtggtcacatctgaatagcagtagct-
ccctaggagaatt
aattccaagttctttggtggtgtatcaatgcccttaaaggggtcaacaactttttttccctctgacaaaactat-
cttcttatgtccttgtccct
catatttgaagtattttattctttgcagtgttgaatatcaattctagcacctcagacatgttaggtaagtaccc-
tacaactcaggttaactaa
tttaatttaactaatttaaccccaacactttttctttgtttatccacatttgtggagtgtgtgtgtgtgtgtgt-
gtgtgtgtgtgtgtgtgtgt
gtgtgtgtgtgtgtgtgtgtgtgtgcgcgcgcgcgcgcgctcggatcattctaccttttgtttaaaaaatgtta-
gtccaggggtggggtgc
actgtgaaagtctgagggtaacttgctggggtcagttattccactataggacagaactccaggtgtcaactatt-
actgacagaacc
atccaaatagccctatctaattttagttttttatttatttattttttgtttttcgagacagggtttctctgtgg-
ctttggaggctgtcctggaa
ctagctatgtagaccaggctggtctcgaactcagagatccacctgcctctgcctcctgagtgctgggattaaag-
gcatgcgccaccaa
cgcttggctctacctaattttaaaagagattgtgtgtcacaagggtgtcatgtcgccctgcaaccacccccccc-
ccaaaaaaaaaaa
aaaaaaaacttcactgaagctgaagcacgatgatttggttactctggctggccaatgagctctagggagtctcc-
tgtcaaacagaat
ctcaacaggcgcagcagtctttttaaagtggggttacaacacaggtttttgcatatcaggcattttatctaagc-
tatttcccagccaaa
aatgtgtattttggaggcagcagagctaatagattaaaatgagggaagagcccacacaggttattaggaagata-
agcatcttctttat
ataaaacaaaaccaaaccaaactggaggaggtctacctttagggatggaagaaaagacatttagagggtgcaat-
agaaagggca
ctgagtttgtgaggtggaggactgggagagggcgcaaccgctttaactgtcctgttttgcctattttttgggga-
cagcacatgttccta
tttttcccaggatgggcaatctccacgtccaaacttgcggtcgaggactacagtcattttgcaggtttccttac-
tgtatggcttttaaaac
gtgcaaaggtgaccattaaccgtttcacgctgggagggcacgtgcggctcagatgatcctctgactgagggcca-
ggagggggc
tacacggaagaggccacacccgcacttgggaagactcgatttgggcttcagctggctgagacgccccagcaggc-
tcctcggcta
caccttcagccccgaatgccttccggcccataaccatccatctaggcatttccggcgaggacccaccctcgcgc-
caaacattcg
gccccatcccccggtcctcacctgaatctctaactctgactccagagtttagagactataaccagatagcccgg-
atgtgtggaactg
catcttgggacgagtagttttagcaaaaagaaagcgacgaaaaactacaattcccagacagacttgtgttacct-
ctcttctcatgctaa
acaagccccctttaaaggaaagcccctcttagtcgcatcgactgtgtaagaaaggcgtttgaaacattttaatg-
ttgggcacaccgttt
cgaggaccgaaatgagaaagagcatagggaaacggagcgcccgagctagtctggcactgcgttagacagccgcg-
g TE ELEMENT 00 (SEQUENCE ID NO. 2)
gatctccaggacagccatggctattacacagagaaaccctgtctggaaaaacaaaaaattagtgtccatgtgta-
aatgtgtggagta
tgcttgtcatgccacatacagaggtagagggcagtttatgggagtcagttcctattcttcctttatgggggacc-
tggggactgaactc
aggtcatcaggcttggcagaaagtgcattagctcacggagccttatcattggcgaaagctctctcaagtagaaa-
atcaatgtgtttgc
tcatagtgcaatcattatgtttcgagaggggaagggtacaatcgttggggcatgtgtggtcacatctgaatagc-
agtagctccctagg
agaattaattccaagttattggtggtgtatcaatgccataaaggggtcaacaactttttttccctctgacaaaa-
ctatcttcttatgtcctt
gtccctcatatttgaagtattttattattgcagtgttgaatatcaattctagcacctcagacatgttaggtaag-
taccctacaactcaggtt
aactaatttaatttaactaatttaaccccaacactttttctttgtttatccacatttgtggagtgtgtgtgtgt-
gtgtgtgtgtgtgtgtgtgt
gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcgcgcgcgcgcgcgctcggatcattctaccttttgtttaaaaa-
atgttagtccaggggtg
gggtgcactgtgaaagtctgagggtaacttgctggggtcagttattccactataggacagaactccaggtgtca-
actattactgac
agaaccatccaaatagccctatctaattttagttttttatttatttattttttgtttttcgagacagggtttct-
ctgtggctttggaggctgtc
ctggaactagctcttgtagaccaggctggtctcgaactcagagatccacctgcctctgcctcctgagtgctggg-
attaaaggcatgcg
ccaccaacgcttggctctacctaattttaaaagagattgtgtgtcacaagggtgtcatgtcgccctgcaaccac-
cccccccccaaaa
aaaaaaaaaaaaaaacttcactgaagctgaagcacgatgatttggttactctggctggccaatgagctctaggg-
agtctcctgtcaa
acagaatctcaacaggcgcagcagtcttttttaaagtggggttacaacacaggtttttgcatatcaggcatttt-
atctaagctatttccca
gccaaaaatgtgtattttggaggcagcagagctaatagattaaaatgagggaagagcccacacaggttattagg-
aagataagcatc
ttctttatataaaacaaaaccaaaccaaactggaggaggtctacctttagggatggaagaaaagacatttagag-
ggtgcaatagaaa
gggcactgagtttgtgaggtggaggactgggagagggcgcaaccgctttaactgtcctgttttgcctatttttt-
ggggacagcacat
gttcctatttttcccaggatgggcaatctccacgtccaaacttgcggtcgaggactacagtcattttgcaggtt-
tccttactgtatggctt
ttaaaacgtgcaaaggtgaccattaaccgtttcacgctgggagggcacgtgcggctcagatgatcctctgactg-
agggccagga
gggggctacacggaagaggccacacccgcacttgggaagactcgatttgggatcagctggctgagacgccccag-
caggctcc
tcggctacaccttcagccccgaatgccttccggcccataaccatcccttctaggcatttccggcgaggacccac-
cctcgcgccaa
acattcggccccatcccccggtcctcacctgaatctctaactctgactccagagtttagagactataaccagat-
agcccggatgtgtg
gaactgcatcttgggacgagtagttttagcaaaaagaaagcgacgaaaaactacaattcccagacagacttgtg-
ttacctctcttctc
atgctaaacaagccccctttaaaggaaagcccctcttagtcgcatcgactgtgtaagaaaggcgtttgaaacat-
tttaatgttgggca
caccgtttcgaggaccgaaatgagaaagagcatagggaaacggagcgcccgagctagtctggcactgcgttaga-
cagccgcgg TE ELEMENT 01 (SEQUENCE ID NO. 3)
gttgctattttagagacaggatttatgcaaacctggttggtcttaaactccgtatgtagctgagaatgaccttg-
aaaaccttcctgtccc
acccctcaaattccagaattatagacacccaccacatggcttaataagtaaacaacaacaataaaagcatgact-
tctgggtctggag
ggagggcttgccagttaagagcaatggatactttcccatagaacctgggtttgactcccagcactaacctacat-
ggtgatagtgatg
cagcagacatacatgagggcaacacacacatgggcacatacacacgcacccgcccaccatggcttttcccccat-
cacttagacag
ccatatttaaacgtagtggagccaggctggggtggtggcccacacctttaatcccagcactccagaaggcagag-
gtaggcggatc
tctgtgggtttgagaccagcctggtctacaagagctagttccaggacagcctccaaagccatagagaaacccta-
tctcaaaaaact
gaaacaacaacaacaacaaaacaaaataaaaaaacaacaaaagaatcttagtggttcagtggttccacacacag-
gaaagtagaaa
gggccttgatgggaaggttttcagagggaggagtatggatgagacaggatgatagtgaaaagaactcaaattaa-
ttaaatatttgaa
actatctaagaataaaagctaaaatatttaaaattacagtcaggtagtggtggtgcagagggctaagttggtag-
acacagtgagatc
caggccagccagggctacctagtgagaccttgttcaaataactaataaaatatacaaaataaaggagacaccac-
aataattttgaaa
tgtaaaagactaaatttaccttttatattgatgagttggataaaaaaatcaatttaccagagaacataaagtag-
tcccatcaaagacaaa
agcaatatatgattaaactctaatttaaaagtttgttagagcctggcaacgtggcacatacctttaatcccagc-
accagggagacaga
ggccatcctggtctaaaaagtgatctccaggacagccatggctattacacagagaaaccctgtctggaaaaaca-
aaaaattagtgt
ccatgtgtaaatgtgtggagtatgatgtcatgccacatacagaggtagagggcagtttatgggagtcagttcct-
attcttcctttatgg
gggacctggggactgaactcaggtcatcaggcttggcagaaagtgcattagctcacggagccttatcattggcg-
aaagctctctca
agtagaaaatcaatgtgtttgctcatagtgcaatcattatgtttcgagaggggaagggtacaatcgttggggca-
tgtgtggtcacatct
gaatagcagtagctccctaggagaattaattccaagttctttggtggtgtatcaatgcccttaaaggggtcaac-
aactttttttccctctg
acaaaactatcttatatgtccttgtccctcatatttgaagtattttattctttgcagtgttgaatatcaattct-
agcacctcagacatgttagg
taagtaccctacaactcaggttaactaatttaatttaactaatttaaccccaacactttttctttgtttatcca-
catttgtggagtgtgtgtgt
gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcgcgcgcgcgcgcgctcggat-
cattctaccttttgtttaa
aaaatgttagtccaggggtggggtgcactgtgaaagtctgagggtaacttgctggggtcagttctttccactat-
aggacagaactcc
aggtgtcaactctttactgacagaaccatccaaatagccctatctaattttagttttttatttatttatttttt-
gtttttcgagacagggtttc
tctgtggcttggaggctgtcctggaactagctcttgtagaccaggctggtctcgaactcagagatccacctgcc-
tctgcctcctgagtg
ctgggattaaaggcatgcgccaccaacgcttggctctacctaattttaaaagagattgtgtgtcacaagggtgt-
catgtcgccctgca
accaccccccccccaaaaaaaaaaaaaaaaaaacttcactgaagctgaagcacgatgatttggttactctggct-
ggccaatgagct
ctagggagtctcctgtcaaacagaatctcaacaggcgcagcagtcttttttaaagtggggttacaacacaggtt-
tttgcatatcaggc
attttatctaagctatttcccagccaaaaatgtgtattttggaggcagcagagctaatagattaaaatgaggga-
agagcccacacagg
ttattaggaagataagcatcttctttatataaaacaaaaccaaaccaaactggaggaggtctacctttagggat-
ggaagaaaagacat
ttagagggtgcaatagaaagggcactgagtttgtgaggtggaggactgggagagggcgcaaccgctttaactgt-
cctgttttgcct
attttttggggacagcacatgttcctatttttcccaggatgggcaatctccacgtccaaacttgcggtcgagga-
ctacagtcattttgca
ggtttccttactgtatggcttttaaaacgtgcaaaggtgaccattaaccgtttcacgctgggagggcacgtgcg-
gctcagatgcttcct
ctgactgagggccaggagggggctacacggaagaggccacacccgcacttgggaagactcgatttgggatcagc-
tggctgag
acgccccagcaggctcctcggctacaccttcagccccgaatgccttccggcccataacccttcccttctaggca-
tttccggcgagg
acccaccctcgcgccaaacattcggccccatcccccggtcctcacctgaatctctaactctgactccagagttt-
agagactataacc agatag TE ELEMENT 02 (SEQUENCE ID NO. 4)
caaagccatagagaaaccctatctcaaaaaactgaaacaacaacaacaacaaaacaaaataaaaaaacaacaaa-
agaatcttagt
ggttcagtggttccacacacaggaaagtagaaagggccttgatgggaaggttttcagagggaggagtatggatg-
agacaggatg
atagtgaaaagaactcaaattaattaaatatttgaaactatctaagaataaaagctaaaatatttaaaattaca-
gtcaggtagtggtggt
gcagagggctaagttggtagacacagtgagatccaggccagccagggctacctagtgagaccttgttcaaataa-
ctaataaaatat
acaaaataaaggagacaccacaataattttgaaatgtaaaagactaaatttaccttttatattgatgagttgga-
taaaaaaatcaatttac
cagagaacataaagtagtcccatcaaagacaaaagcaatatatgattaaactctaatttaaaagtttgttagag-
cctggcaacgtggc
acatacctttaatcccagcaccagggagacagaggccatcctggtctaaaaagtgatctccaggacagccatgg-
ctattacacaga
gaaaccctgtctggaaaaacaaaaaattagtgtccatgtgtaaatgtgtggagtatgcttgtcatgccacatac-
agaggtagagggc
agtttatgggagtcagttcctattatcctttatgggggacctggggactgaactcaggtcatcaggcttggcag-
aaagtgcattagct
cacggagccttatcattggcgaaagctctctcaagtagaaaatcaatgtgtttgctcatagtgcaatcattatg-
tttcgagaggggaag
ggtacaatcgttggggcatgtgtggtcacatctgaatagcagtagctccctaggagaattaattccaagttctt-
tggtggtgtatcaat
gcccttaaaggggtcaacaactttttttccctctgacaaaactatcttcttatgtccttgtccctcatatttga-
agtattttattctttgcagt
gttgaatatcaattctagcacctcagacatgttaggtaagtaccctacaactcaggttaactaatttaatttaa-
ctaatttaaccccaacact
ttttattgtttatccacatttgtggagtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt-
gtgtgtgtgtgtgtgcgcg
cgcgcgcgcgctcggatcattctaccttttgtttaaaaaatgttagtccaggggtggggtgcactgtgaaagtc-
tgagggtaacttgc
tggggtcagttctttccactataggacagaactccaggtgtcaactctttactgacagaaccatccaaatagcc-
ctatctaattttagttt
tttatttatttattttttgtttttcgagacagggtttctctgtggctttggaggctgtcctggaactagctctt-
gtagaccaggctggtctcga
actcagagatccacctgcctctgcctcctgagtgctgggattaaaggcatgcgccaccaacgcttggctctacc-
taattttaaaaga
gattgtgtgtcacaagggtgtcatgtcgccctgcaaccaccccccccccaaaaaaaaaaaaaaaaaaacttcac-
tgaagctgaag
cacgatgatttggttactctggctggccaatgagctctagggagtctcctgtcaaacagaatctcaacaggcgc-
agcagtcttttttaa
agtggggttacaacacaggtttttgcatatcaggcattttatctaagctatttcccagccaaaaatgtgtattt-
tggaggcagcagagct
aatagattaaaatgagggaagagcccacacaggttattaggaagataagcatcttattatataaaacaaaacca-
aaccaaactgga
ggaggtctacctttagggatggaagaaaagacatttagagggtgcaatagaaagggcactgagtttgtgaggtg-
gaggactggga
gagggcgcaaccgctttaactgtcctgttttgcctattttttggggacagcacatgttcctatttttcccagga-
tgggcaatctccacgtc
caaacttgcggtcgaggactacagtcattttgcaggtttccttactgtatggcttttaaaacgtgcaaaggtga-
ccattaaccgtttcac
gctgggagggcacgtgcggctcagatgcttcctctgactgagggccaggagggggctacacggaagaggccaca-
cccgcactt
gggaagactcgatttgggatcagctggctgagacgccccagcaggctcctcggctacaccttcagccccgaatg-
ccttccggcc
cataacccttcccttctaggcatttccggcgaggacccaccctcgcgccaaacattcggccccatcccccggtc-
ctcacctgaatct ctaactctgactccagagtttagagactataaccagatag TE ELEMENT
03 (SEQUENCE ID NO. 5)
acctttaatcccagcaccagggagacagaggccatcctggtctaaaaagtgatctccaggacagccatggctat-
tacacagagaa
accctgtctggaaaaacaaaaaattagtgtccatgtgtaaatgtgtggagtatgcttgtcatgccacatacaga-
ggtagagggcagtt
tatgggagtcagttcctattcttcctttatgggggacctggggactgaactcaggtcatcaggcttggcagaaa-
gtgcattagctcac
ggagccttatcattggcgaaagctctctcaagtagaaaatcaatgtgtttgctcatagtgcaatcattatgttt-
cgagaggggaagggt
acaatcgttggggcatgtgtggtcacatctgaatagcagtagctccctaggagaattaattccaagttctttgg-
tggtgtatcaatgcc
cttaaaggggtcaacaactttttttccctctgacaaaactatcttcttatgtccttgtccctcatatttgaagt-
attttattctttgcagtgtt
gaatatcaattctagcacctcagacatgttaggtaagtaccctacaactcaggttaactaatttaatttaacta-
atttaaccccaacacttttt
ctttgtttatccacatttgtggagtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtg-
tgtgtgtgtgtgcgcgcgcg
cgcgcgctcggatcattctaccttttgtttaaaaaatgttagtccaggggtggggtgcactgtgaaagtctgag-
ggtaacttgctggg
gtcagttattccactataggacagaactccaggtgtcaactctttactgacagaaccatccaaatagccctatc-
taattttagttttttatt
tatttattttttgtttttcgagacagggtttctctgtggctttggaggctgtcctggaactagctcttgtagac-
caggctggtctcgaactc
agagatccacctgcctctgcctcctgagtgctgggattaaaggcatgcgccaccaacgcttggctctacctaat-
tttaaaagagattg
tgtgtcacaagggtgtcatgtcgccctgcaaccaccccccccccaaaaaaaaaaaaaaaaaaacttcactgaag-
ctgaagcacga
tgatttggttactctggctggccaatgagctctagggagtctcctgtcaaacagaatctcaacaggcgcagcag-
tcttttttaaagtgg
ggttacaacacaggtttttgcatatcaggcattttatctaagctatttcccagccaaaaatgtgtattttggag-
gcagcagagctaatag
attaaaatgagggaagagcccacacaggttattaggaagataagcatcttctttatataaaacaaaaccaaacc-
aaactggaggag
gtctacctttagggatggaagaaaagacatttagagggtgcaatagaaagggcactgagtttgtgaggtggagg-
actgggagagg
gcgcaaccgctttaactgtcctgttttgcctattttttggggacagcacatgttcctatttttcccaggatggg-
caatctccacgtccaaa
cttgcggtcgaggactacagtcattttgcaggtttccttactgtatggcttttaaaacgtgcaaaggtgaccat-
taaccgtttcacgctg
ggagggcacgtgcggctcagatgcttcctctgactgagggccaggagggggctacacggaagaggccacacccg-
cacttggg
aagactcgatttgggatcagctggctgagacgccccagcaggctcctcggctacaccttcagccccgaatgcct-
tccggcccata
acccttcccttctaggcatttccggcgaggacccaccctcgcgccaaacattcggccccatcccccggtcctca-
cctgaatctctaa ctctgactccagagtttagagactataaccagatag TE ELEMENT 04
(SEQUENCE ID NO. 6)
ctatcttatatgtccttgtccctcatatttgaagtattttattctttgcagtgttgaatatcaattctagcacc-
tcagacatgttaggtaagta
ccctacaactcaggttaactaatttaatttaactaatttaaccccaacacttttttctttgtttatccacattt-
gtggagtgtgtgtgtgtgtg
tgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcgcgcgcgcgcgcgctcggatcattc-
taccttttgtttaaaaaat
gttagtccaggggtggggtgcactgtgaaagtctgagggtaacttgctggggtcagttctttccactataggac-
agaactccaggtg
tcaactctttactgacagaaccatccaaatagccctatctaattttagttttttatttatttattttttgtttt-
tcgagacagggtttctctgt
ggctttggaggctgtcctggaactagctcttgtagaccaggctggtctcgaactcagagatccacctgcctctg-
cctcctgagtgctggg
attaaaggcatgcgccaccaacgcttggctctacctaattttaaaagagattgtgtgtcacaagggtgtcatgt-
cgccctgcaaccac
cccccccccaaaaaaaaaaaaaaaaaaacttcactgaagctgaagcacgatgatttggttactctggctggcca-
atgagctctagg
gagtctcctgtcaaacagaatctcaacaggcgcagcagtcttttttaaagtggggttacaacacaggtttttgc-
atatcaggcattttat
ctaagctatttcccagccaaaaatgtgtattttggaggcagcagagctaatagattaaaatgagggaagagccc-
acacaggttatta
ggaagataagcatcttctttatataaaacaaaaccaaaccaaactggaggaggtctacctttagggatggaaga-
aaagacatttaga
gggtgcaatagaaagggcactgagtttgtgaggtggaggactgggagagggcgcaaccgctttaactgtcctgt-
tttgcctatttttt
ggggacagcacatgttcctatttttcccaggatgggcaatctccacgtccaaacttgcggtcgaggactacagt-
cattttgcaggtttc
cttactgtatggattttaaaacgtgcaaaggtgaccattaaccgtttcacgctgggagggcacgtgcggctcag-
atgcttcctctgac
tgagggccaggagggggctacacggaagaggccacacccgcacttgggaagactcgatttgggcttcagctggc-
tgagacgcc
ccagcaggctcctcggctacaccttcagccccgaatgccttccggcccataacccttcccttctaggcatttcc-
ggcgaggaccca
ccctcgcgccaaacattcggccccatcccccggtcctcacctgaatctctaactctgactccagagtttagaga-
ctataaccagatag TE ELEMENT 05 (SEQUENCE ID NO. 7)
caggctggtctcgaactcagagatccacctgcctctgcctcctgagtgctgggattaaaggcatgcgccaccaa-
cgcttggctcta
cctaattttaaaagagattgtgtgtcacaagggtgtcatgtcgccctgcaaccaccccccccccaaaaaaaaaa-
aaaaaaaaacttc
actgaagctgaagcacgatgatttggttactctggctggccaatgagctctagggagtctcctgtcaaacagaa-
tctcaacaggcgc
agcagtcttttttaaagtggggttacaacacaggtttttgcatatcaggcattttatctaagctatttcccagc-
caaaaatgtgtattttgg
aggcagcagagctaatagattaaaatgagggaagagcccacacaggttattaggaagataagcatcttctttat-
ataaaacaaaac
caaaccaaactggaggaggtctacctttagggatggaagaaaagacatttagagggtgcaatagaaagggcact-
gagtttgtgag
gtggaggactgggagagggcgcaaccgctttaactgtcctgttttgcctattttttggggacagcacatgttcc-
tatttttcccaggatg
ggcaatctccacgtccaaacttgcggtcgaggactacagtcattttgcaggtttccttactgtatggcttttaa-
aacgtgcaaaggtga
ccattaaccgtttcacgctgggagggcacgtgcggctcagatgatcctctgactgagggccaggagggggctac-
acggaagag
gccacacccgcacttgggaagactcgatttgggcttcagctggctgagacgccccagcaggctcctcggctaca-
ccttcagcccc
gaatgccttccggcccataacccttcccttctaggcatttccggcgaggacccaccctcgcgccaaacattcgg-
ccccatcccccg
gtcctcacctgaatctctaactctgactccagagtttagagactataaccagatag TE ELEMENT
06 (SEQUENCE ID NO. 8)
Cttgcggtcgaggactacagtcattttgcaggtttccttactgtatggcttttaaaacgtgcaaaggtgaccat-
taaccgtttcacgctg
ggagggcacgtgcggctcagatgcttcctctgactgagggccaggagggggctacacggaagaggccacacccg-
cacttggg
aagactcgatttgggcttcagctggctgagacgccccagcaggctcctcggctacaccttcagccccgaatgcc-
ttccggcccata
acccttcccttctaggcatttccggcgaggacccaccctcgcgccaaacattcggccccatcccccggtcctca-
cctgaatctctaa ctctgactccagagtttagcgactataaccagatag TE ELEMENT 07
(SEQUENCE ID NO. 9)
Gcctgaagacctgagttgatacccagaacccagatcaagatggaggagagaaccagccccactaagctgtcccc-
tgacccccat
aaatgcctccctgtccagttatgccacacaatgataggtgaatacagaaaaacacccttcctttagacactaag-
cggattcctcttac
gcataccagttaagtgatagttcttaggcttcaactcagcactttaaaaagtttatattttgcaatgctgggga-
ctaaattagggttgtgc
acatgctaagtaagcactctacttttgtatcacattttaataattgtaagaattaattcgtgaaatagtagctg-
agacaatagatttgtttctt
tcatgtgggaactgctgtgtgtgcttcttgctgatgcaaacaaggtcaaatactttattccccagtgtctgcct-
agccctgtaacacttct
ctattatacaatgaccacaaataattaggtgagtgggttttgtttcattttaaattgttgctattttagagaca-
ggatttc TE ELEMENT 08 (SEQUENCE ID NO. 10)
Gcctgaagacctgagttgatacccagaacccagatcaagatggaggagagaaccagccccactaagctgtcccc-
tgacccccat
aaatgcctccctgtccagttatgccacacaatgataggtgaatacagaaaaacacccttcctttagacactaag-
cggattcctcttac
gcataccagttaagtgatagttcttaggcttcaactcagcactttaaaaagtttatattttgcaatgctgggga-
ctaaattagggttgtgc
acatgctaagtaagcactctacttttgtatcacattttaataattgtaagaattaattcgtgaaatagtagctg-
agacaatagatttgtttctt
tcatgtgggaactgctgtgtgtgcttcttgctgatgcaaacaaggtcaaatactttattccccagtgtctgcct-
agccctgtaacacttct
ctattatacaatgaccacaaataattaggtgagtgggttttgtttcattttaaattgttgctattttagagaca-
ggatttcttgcaaacctggt
tggtcttaaactccgtatgtagctgagaatgaccttgaaaaccttcctgtcccacccctcaaattccagaatta-
tagacacccaccaca
tggcttaataagtaaacaacaacaataaaagcatgacttctgggtctggagggagggcttgccagttaagagca-
atggatactttcc
catagaacctgggtttgactcccagcactaacctacatggtgatagtgatgcagcagacatacatgagggcaac-
acacacatggg
cacatacacacgcacccgcccaccatggcttttcccccatcacttagacagccatatttaaacgtagtggagcc-
aggctggggtgg
tggcccacacctttaatcccagcactccagaaggcagaggtaggcggatctctgtgggtttgagaccagcctgg-
tctacaagagct agttccaggacagcctccaaagccatagagaaaccctatc TE ELEMENT 09
(SEQUENCE ID NO. 11)
gcctgaagacctgagttgatacccagaacccagatcaagatggaggagagaaccagccccactaagctgtcccc-
tgacccccat
aaatgcctccctgtccagttatgccacacaatgataggtgaatacagaaaaacacccttcctttagacactaag-
cggattcctcttac
gcataccagttaagtgatagttcttaggcttcaactcagcactttaaaaagtttatattttgcaatgctgggga-
ctaaattagggttgtgc
acatgctaagtaagcactctacttttgtatcacattttaataattgtaagaattaattcgtgaaatagtagctg-
agacaatagatttgtttctt
tcatgtgggaactgctgtgtgtgcttcttgctgatgcaaacaaggtcaaatactttattccccagtgtctgcct-
agccctgtaacacttct
ctattatacaatgaccacaaataattaggtgagtgggttttgtttcattttaaattgttgctattttagagaca-
ggatttcttgcaaacctggt
tggtcttaaactccgtatgtagctgagaatgaccttgaaaaccttcctgtcccacccctcaaattccagaatta-
tagacacccaccaca
tggcttaataagtaaacaacaacaataaaagcatgacttctgggtctggagggagggcttgccagttaagagca-
atggatactttcc
catagaacctgggtttgactcccagcactaacctacatggtgatagtgatgcagcagacatacatgagggcaac-
acacacatggg
cacatacacacgcacccgcccaccatggcttttcccccatcacttagacagccatatttaaacgtagtggagcc-
aggctggggtgg
tggcccacacctttaatcccagcactccagaaggcagaggtaggcggatctctgtgggtttgagaccagcctgg-
tctacaagagct
agttccaggacagcctccaaagccatagagaaaccctatctcaaaaaactgaaacaacaacaacaacaaaacaa-
aataaaaaaa
caacaaaagaatcttagtggttcagtggttccacacacaggaaagtagaaagggccttgatgggaaggttttca-
gagggaggagt
atggatgagacaggatgatagtgaaaagaactcaaattaattaaatatttgaaactatctaagaataaaagcta-
aaatatttaaaattac
agtcaggtagtggtggtgcagagggctaagttggtagacacagtgagatccaggccagccagggctacctagtg-
agaccttgttc
aaataactaataaaatatacaaaataaaggagacaccacaataattttgaaatgtaaaagactaaatttacctt-
ttatattgatgagttgg
ataaaaaaatcaatttaccagagaacataaagtagtcccatcaaagacaaaagcaatatatgattaaactctaa-
tttaaaagtttgttag agcctggcaacgtggcacatacctttaatcccagcaccagg TE
ELEMENT 10 (SEQUENCE ID NO. 12)
gcctgaagacctgagttgatacccagaacccagatcaagatggaggagagaaccagccccactaagctgtcccc-
tgacccccat
aaatgcctccctgtccagttatgccacacaatgataggtgaatacagaaaaacacccttcctttagacactaag-
cggattcctcttac
gcataccagttaagtgatagttcttaggcttcaactcagcactttaaaaagtttatattttgcaatgctgggga-
ctaaattagggttgtgc
acatgctaagtaagcactctacttttgtatcacattttaataattgtaagaattaattcgtgaaatagtagctg-
agacaatagatttgtttctt
tcatgtgggaactgctgtgtgtgcttcttgctgatgcaaacaaggtcaaatactttattccccagtgtctgcct-
agccctgtaacacttct
ctattatacaatgaccacaaataattaggtgagtgggttttgtttcattttaaattgttgctattttagagaca-
ggatttcttgcaaacctggt
tggtcttaaactccgtatgtagctgagaatgaccttgaaaaccttcctgtcccacccctcaaattccagaatta-
tagacacccaccaca
tggcttaataagtaaacaacaacaataaaagcatgacttctgggtctggagggagggcttgccagttaagagca-
atggatactttcc
catagaacctgggtttgactcccagcactaacctacatggtgatagtgatgcagcagacatacatgagggcaac-
acacacatggg
cacatacacacgcacccgcccaccatggcttttcccccatcacttagacagccatatttaaacgtagtggagcc-
aggctggggtgg
tggcccacacctttaatcccagcactccagaaggcagaggtaggcggatctctgtgggtttgagaccagcctgg-
tctacaagagct
agttccaggacagcctccaaagccatagagaaaccctatctcaaaaaactgaaacaacaacaacaacaaaacaa-
aataaaaaaa
caacaaaagaatcttagtggttcagtggttccacacacaggaaagtagaaagggccttgatgggaaggttttca-
gagggaggagt
atggatgagacaggatgatagtgaaaagaactcaaattaattaaatatttgaaactatctaagaataaaagcta-
aaatatttaaaattac
agtcaggtagtggtggtgcagagggctaagttggtagacacagtgagatccaggccagccagggctacctagtg-
agaccttgttc
aaataactaataaaatatacaaaataaaggagacaccacaataattttgaaatgtaaaagactaaatttacctt-
ttatattgatgagttgg
ataaaaaaatcaatttaccagagaacataaagtagtcccatcaaagacaaaagcaatatatgattaaactctaa-
tttaaaagtttgttag
agcctggcaacgtggcacatacctttaatcccagcaccagggagacagaggccatcctggtctaaaaagtgatc-
tccaggacagc
catggctattacacagagaaaccctgtctggaaaaacaaaaaattagtgtccatgtgtaaatgtgtggagtatg-
cttgtcatgccacat
acagaggtagagggcagtttatgggagtcagttcctattcttcctttatgggggacctggggactgaactcagg-
tcatcaggcttgg
cagaaagtgcattagctcacggagccttatcattggcgaaagctctctcaagtagaaaatcaatgtgtttgctc-
atagtgcaatcatta
tgtttcgagaggggaagggtacaatcgttggggcatgtgtggtcacatctgaatagcagtagctccctaggaga-
attaattccaagtt
ctttggtggtgtatcaatgcccttaaaggggtcaacaactttttttccctctgacaaaactatcttcttatgtc-
cttgtccc TE ELEMENT 11 (SEQUENCE ID NO. 13)
gcctgaagacctgagttgatacccagaacccagatcaagatggaggagagaaccagccccactaagctgtcccc-
tgacccccat
aaatgcctccctgtccagttatgccacacaatgataggtgaatacagaaaaacacccttcctttagacactaag-
cggattcctcttac
gcataccagttaagtgatagttcttaggcttcaactcagcactttaaaaagtttatattttgcaatgctgggga-
ctaaattagggttgtgc
acatgctaagtaagcactctacttttgtatcacattttaataattgtaagaattaattcgtgaaatagtagctg-
agacaatagatttgtttctt
tcatgtgggaactgctgtgtgtgcttcttgctgatgcaaacaaggtcaaatactttattccccagtgtctgcct-
agccctgtaacacttct
ctattatacaatgaccacaaataattaggtgagtgggttttgtttcattttaaattgttgctattttagagaca-
ggatttcttgcaaacctggt
tggtcttaaactccgtatgtagctgagaatgaccttgaaaaccttcctgtcccacccctcaaattccagaatta-
tagacacccaccaca
tggcttaataagtaaacaacaacaataaaagcatgacttctgggtctggagggagggcttgccagttaagagca-
atggatactttcc
catagaacctgggtttgactcccagcactaacctacatggtgatagtgatgcagcagacatacatgagggcaac-
acacacatggg
cacatacacacgcacccgcccaccatggatttcccccatcacttagacagccatatttaaacgtagtggagcca-
ggctggggtgg
tggcccacacctttaatcccagcactccagaaggcagaggtaggcggatctctgtgggtttgagaccagcctgg-
tctacaagagct
agttccaggacagcctccaaagccatagagaaaccctatctcaaaaaactgaaacaacaacaacaacaaaacaa-
aataaaaaaa
caacaaaagaatcttagtggttcagtggttccacacacaggaaagtagaaagggccttgatgggaaggttttca-
gagggaggagt
atggatgagacaggatgatagtgaaaagaactcaaattaattaaatatttgaaactatctaagaataaaagcta-
aaatatttaaaattac
agtcaggtagtggtggtgcagagggctaagttggtagacacagtgagatccaggccagccagggctacctagtg-
agaccttgttc
aaataactaataaaatatacaaaataaaggagacaccacaataattttgaaatgtaaaagactaaatttacctt-
ttatattgatgagttgg
ataaaaaaatcaatttaccagagaacataaagtagtcccatcaaagacaaaagcaatatatgattaaactctaa-
tttaaaagtttgttag
agcctggcaacgtggcacatacctttaatcccagcaccagggagacagaggccatcctggtctaaaaagtgatc-
tccaggacagc
catggctattacacagagaaaccctgtctggaaaaacaaaaaattagtgtccatgtgtaaatgtgtggagtatg-
cttgtcatgccacat
acagaggtagagggcagtttatgggagtcagttcctattcttcctttatgggggacctggggactgaactcagg-
tcatcaggcttgg
cagaaagtgcattagctcacggagccttatcattggcgaaagctctctcaagtagaaaatcaatgtgtttgctc-
atagtgcaatcatta
tgtttcgagaggggaagggtacaatcgttggggcatgtgtggtcacatctgaatagcagtagctccctaggaga-
attaattccaagtt
ctttggtggtgtatcaatgcccttaaaggggtcaacaactttttttccctctgacaaaactatcttcttatgtc-
cttgtccctcatatttgaag
tattttattctttgcagtgttgaatatcaattctagcacctcagacatgttaggtaagtaccctacaactcagg-
ttaactaatttaatttaact
aatttaaccccaacactttttctttgtttatccacatttgtggagtgtgtgtgtgtgtgtgtgtgtgtgtgtgt-
gtgtgtgtgtgtgtgtgtgt
gtgtgtgtgtgtgcgcgcgcgcgcgcgctcggatcattctaccttttgtttaaaaaatgttagtccaggggtgg-
ggtgcactgtgaaag
tctgagggtaacttgctggggtcagttctttccactataggacagaactccaggtgtcaactctttactgacag-
aaccatccaaatagc
cctatctaattttagttttttatttatttattttttgtttttcgagacagggtttctctgtggctttggaggct-
gtcctggaactagctcttgt agaccaggctggtctcgaactcag TE ELEMENT 12
(SEQUENCE ID NO. 14)
gcctgaagacctgagttgatacccagaacccagatcaagatggaggagagaaccagccccactaagctgtcccc-
tgacccccat
aaatgcctccctgtccagttatgccacacaatgataggtgaatacagaaaaacacccttcctttagacactaag-
cggattcctcttac
gcataccagttaagtgatagttcttaggcttcaactcagcactttaaaaagtttatattttgcaatgctgggga-
ctaaattagggttgtgc
acatgctaagtaagcactctacttttgtatcacattttaataattgtaagaattaattcgtgaaatagtagctg-
agacaatagatttgtttctt
tcatgtgggaactgctgtgtgtgcttcttgctgatgcaaacaaggtcaaatactttattccccagtgtctgcct-
agccctgtaacacttct
ctattatacaatgaccacaaataattaggtgagtgggttttgtttcattttaaattgttgctattttagagaca-
ggatttcttgcaaacctggt
tggtcttaaactccgtatgtagctgagaatgaccttgaaaaccttcctgtcccacccctcaaattccagaatta-
tagacacccaccaca
tggcttaataagtaaacaacaacaataaaagcatgacttctgggtctggagggagggcttgccagttaagagca-
atggatactttcc
catagaacctgggtttgactcccagcactaacctacatggtgatagtgatgcagcagacatacatgagggcaac-
acacacatggg
cacatacacacgcacccgcccaccatggcttttcccccatcacttagacagccatatttaaacgtagtggagcc-
aggctggggtgg
tggcccacacctttaatcccagcactccagaaggcagaggtaggcggatctctgtgggtttgagaccagcctgg-
tctacaagagct
agttccaggacagcctccaaagccatagagaaaccctatctcaaaaaactgaaacaacaacaacaacaaaacaa-
aataaaaaaa
caacaaaagaatcttagtggttcagtggttccacacacaggaaagtagaaagggccttgatgggaaggttttca-
gagggaggagt
atggatgagacaggatgatagtgaaaagaactcaaattaattaaatatttgaaactatctaagaataaaagcta-
aaatatttaaaattac
agtcaggtagtggtggtgcagagggctaagttggtagacacagtgagatccaggccagccagggctacctagtg-
agaccttgttc
aaataactaataaaatatacaaaataaaggagacaccacaataattttgaaatgtaaaagactaaatttacctt-
ttatattgatgagttgg
ataaaaaaatcaatttaccagagaacataaagtagtcccatcaaagacaaaagcaatatatgattaaactctaa-
tttaaaagtttgttag
agcctggcaacgtggcacatacctttaatcccagcaccagggagacagaggccatcctggtctaaaaagtgatc-
tccaggacagc
catggctattacacagagaaaccctgtctggaaaaacaaaaaattagtgtccatgtgtaaatgtgtggagtatg-
cttgtcatgccacat
acagaggtagagggcagtttatgggagtcagttcctattcttcctttatgggggacctggggactgaactcagg-
tcatcaggcttgg
cagaaagtgcattagctcacggagccttatcattggcgaaagctctctcaagtagaaaatcaatgtgtttgctc-
atagtgcaatcatta
tgtttcgagaggggaagggtacaatcgttggggcatgtgtggtcacatctgaatagcagtagctccctaggaga-
attaattccaagtt
ctttggtggtgtatcaatgcccttaaaggggtcaacaactttttttccctctgacaaaactatcttcttatgtc-
cttgtccctcatatttgaag
tattttattctttgcagtgttgaatatcaattctagcacctcagacatgttaggtaagtaccctacaactcagg-
ttaactaatttaatttaact
aatttaaccccaacactttttctttgtttatccacatttgtggagtgtgtgtgtgtgtgtgtgtgtgtgtgtgt-
gtgtgtgtgtgtgtgtgtgtg
tgtgtgtgtgtgcgcgcgcgcgcgcgctcggatcattctaccttttgtttaaaaaatgttagtccaggggtggg-
gtgcactgtgaaag
tctgagggtaacttgctggggtcagttctttccactataggacagaactccaggtgtcaactctttactgacag-
aaccatccaaatagc
cctatctaattttagttttttatttatttattttttgtttttcgagacagggtttctctgtggctttggaggct-
gtcctggaactagctcttgt
agaccaggctggtctcgaactcagagatccacctgcctctgcctcctgagtgctgggattaaaggcatgcgcca-
ccaacgcttggctct
acctaattttaaaagagattgtgtgtcacaagggtgtcatgtcgccctgcaaccaccccccccccaaaaaaaaa-
aaaaaaaaaactt
cactgaagctgaagcacgatgatttggttactctggctggccaatgagctctagggagtctcctgtcaaacaga-
atctcaacaggcg
cagcagtcttttttaaagtggggttacaacacaggtttttgcatatcaggcattttatctaagctatttcccag-
ccaaaaatgtgtattttg
gaggcagcagagctaatagattaaaatgagggaagagcccacacaggttattaggaagataagcatcttcttta-
tataaaacaaaa
ccaaaccaaactggaggaggtctacctttagggatggaagaaaagacatttagagggtgcaatagaaagggcac-
tgagtttgtga
ggtggaggactgggagagggcgcaaccgctttaactgtcctgttttgcctattttttggggacagcacatgttc-
ctatttttcccaggat gggcaatctccacgtccaaacttgcggtcgaggactacag
Example 3
Influence of the TE Element Variant TE-00 on the Expression of GFP
and Immunoglobulin G1 (IgG1)
[0290] The effect of the TE element TE-00 on the expression of the
cytoplasmically located GFPs (green fluorescent protein) and a
secreted monoclonal IgG1-antibody was investigated in two
independent stable transfection series with CHO-DG44 cells. For
this, CHO-DG44 cells were co-transfected with the following plasmid
combinations or plasmid variants:
control plasmids pBING-LC (FIG. 1A) and pBID-HC (FIG. 1A) without
TE element pBING-LC and pBID-HC with a TE element TE-00 integrated
upstream from the promoter/enhancer in direct orientation pBING-LC
and pBID-HC with a TE element TE-00 integrated upstream from the
promoter/enhancer in reverse orientation
[0291] In transfection series A four pools were produced, in
transfection series B ten pools were produced per variant.
Equimolar amounts of the two plasmids were used. In order to arrive
at the same total number of molecules, the total amount of DNA used
in series A in the control mixtures was 1 .mu.g, while in the
mixtures containing TE element the amount was 1.3 .mu.g. This
difference resulted from the different plasmid sizes, as the
plasmids with TE element were larger than the control plasmids by a
factor of 1.3. As the amount of DNA used in the transfection mix
can have an effect on transfection efficiency, in series B the
total amount of DNA was balanced out with 300 ng of "mock DNA"
(=vector without product gene, TE element and eukaryotic selectable
marker), so that the mixture with the control plasmids also
contained 1.3 .mu.g DNA in total. As a negative control, a
mock-transfected pool was also run in each transfection series,
i.e. treated in the same way, but without the addition of DNA in
the transfection mixture. The selection of stably transfected cells
took place two days after the transfection with -HT/+G418 (400
.mu.g/mL). After the selection the proportion of GFP-expressing
cells was determined by FACS. The comparison of the variants in the
plot overlay in both transfection series yielded a larger
proportion of GFP-expressing cells for pools with TE element 00
than in pools with control plasmids (FIG. 7). Between the pools in
which the TE element was present in the plasmid in either direct or
reverse orientation, no differences of any kind could be found. The
effect of the TE element 00, namely increasing the proportion of
cells with higher productivity in a mixed population, was
consequently independent of the orientation thereof.
[0292] In addition, the IgG1 titre and the specific productivity of
the pools were determined over a period of six to eight passages
(passaging rhythm 2-2-3 days). Here again it was confirmed that the
cell pools containing the TE element 00 on average expressed more
than the cell pools without TE element (FIG. 9, series A and B). In
both series, a doubling of the pool productivity could be
demonstrated as a result of the presence of the TE element, while
the orientation in which the element was cloned in the expression
plasmid was of no relevance.
Example 4
Influence of the TE Elements TE-01 to TE-12 on the Expression of
MCP-1
[0293] The effect of the TE elements TE-01 to TE-12 on the
expression of the secreted MCP-1 was investigated in three stable
transfection series (Series C, D and E) of CHO-DG44 cells compared
with expression without the TE element. In all three series, 6
pools were produced per plasmid variant. The base plasmid was
pTE4/MCP-1 in Series C and D(FIG. 1B; Selectable Marker
NPT--Neomycin-phosphotransferase F240I), pTE5/MCP-1 in Series E
(FIG. 2; Selectable Marker DHFR=Dihydrofolate-reductase). These
contained either no TE element (=control mixtures) or one of the TE
elements TE-01 to TE-12 in direct orientation upstream of the
promoter/enhancer. In order to minimise the influence on
transfection efficiency caused by different amounts of DNA in the
transfection mixture, 1.2 .mu.g of plasmid-DNA were used in total.
Depending on the size of the TE element introduced, the plasmid
size varied between 6.7 kb and 10.7 kb. However, to ensure that the
total number of molecule of test plasmids could be kept constant in
all the mixtures, in the mixtures with smaller plasmid molecules
the total amount of DNA was balanced out with a so-called mock
plasmid which contained neither product gene and TE element nor any
eukaryotic selectable marker. As a negative control, for each
transfection series, a mock-transfected pool was also run, i.e.
treated in the same way but without the addition of DNA to the
transfection mixture. The selection of stably transfected cells was
carried out two days after transfection, with HT-supplemented
CHO-S-SFMII+G418 (400 .mu.g/mL) in Series C and D and with HT-free
CHO-S-SFMII in Series E.
[0294] After the selection, the proportion of dsRed2-expressing
cells was determined by FACS. FIG. 8 shows the relative percentage
fluorescence of the living transfected cells from Series C.
Compared with the control pools, the pools which contained the TE
elements TE-01, TE-02 or TE-08, contained about 3 to 3.5 times more
dsRed2-expressing cells and pools with the element TE-06 contained
approximately twice as many dsRed2-expressing cells. In pools with
the fragments TE-05 and TE-09, on the other hand, there was no
apparent increase in the proportion of dsRed2-expressing cells
compared with the control.
[0295] In addition, the MCP-1 product titre and the specific
productivity were also raised over a period of 6 passages
(passaging rhythm 2-2-3 days). FIG. 9 (Series C and D) and FIG. 10
(Series E) shows a relative specific MCP-1 productivities. The
element TE-08 in conjunction with NPT-F240I as selectable marker
with factor 5.3 showed the greatest increase in the specific MCP-1
productivity compared with the control pools without TE element
(FIG. 9). Combined with DHFR as selectable marker, a 6-fold
increase was achieved with this variant (FIG. 10). The TE elements
01, 02 and 03 resulted in a 4 to 4.5-fold increase in productivity
in the NPT-selected pools (FIG. 9) and a 2.6 to 6.8-fold increase
in productivity in the DHFR-selected pools (FIG. 10) compared with
the control pools. The TE element 06 which is only 300 by long was
also able to increase productivity in all the series by a factor
2.5 to 3.2 (FIGS. 9 and 10). The increases achieved with fragments
TE-04 and TE-07 were also of this order of magnitude (FIG. 10).
Pools in which the somewhat longer fragments TE-10, TE-11 and TE-12
were used, showed a doubling of MCP-1 expression (FIGS. 9 and 10).
Obviously, in all these pools, the number of cells expressing
little or no product was reduced and thus overall the proportion of
high producers in the cell population was increased. This is an
indication that the TE elements are able to suppress, shield or
cancel out negative chromosomal positional effects.
[0296] By contrast, the expression could not be increased compared
with the control by the use of fragments TE-05 and TE-09, as has
already been seen with dsRed2-expression (FIG. 8), and in some
cases was even less (FIGS. 9 and 10). These elements, or partial
fragments in these sequence regions, could possibly thus even have
a repressing effect.
[0297] In all, the change in MCP-1 expression observed correlated
with the proportion of dsRed2-expressing cells in the stable cell
pools.
Example 5
Test of the TE Elements TE-01 to TE-12 on Enhancer Activity
[0298] By transient transfection of CHO-DG44 cells a test was
carried out to see whether the observed increase in product
expression is actually based on a chromatin-opening effect of the
TE elements or whether it is based on an enhancer activity. As the
plasmid is not integrated into the genome in transient
transfection, the genetic information is read off directly from the
plasmid. Thus, no chromosomal positional effects can occur. If
nevertheless there are positive effects on gene expression these
can be put down to enhancers present in the TE element. Such
enhancers can act on the activity of a promoter in the cis location
irrespective of position and orientation and stimulate the
transcription of a functionally linked gene.
[0299] In the transient expression study shown in FIG. 11 6 pools
were transfected with the base plasmid pTE4/MCP-1 (=control; FIG.
1B) or derivatives thereof, each additionally containing one of the
TE elements TE-01 to TE-12 upstream of the promoter/enhancer. After
48 hours cultivation in a total volume of 3 mL harvesting and
determination of the MCP-1 titre were carried out in the cell
culture supernatant by ELISA. Differences in transfection
efficiency were corrected by co-transfection with an SEAP
expression plasmid (addition of 100 ng of plasmid DNA per
transfection mixture) and subsequent measurement of the SEAP
activity. FIG. 11 shows the average from the 6 parallel pools. The
data show that the MCP-1 titre in the cell culture supernatant were
very similar in all the pools and there were no significant
differences in expression from the control plasmid pTE4/MCP-1
without a TE element. Thus the increase on productivity of more
than factor 2 brought about by some TE elements in stably
transfected cell pools is not based on the presence of an enhancer
in the TE sequence. Thus for the enhanced expression obtained by TE
elements chromosomal integration is absolutely essential.
Example 6
Production of Other TE Elements and Testing of Different TE Element
Positions and Combinations
[0300] Analogously to the method described in Example 2, other
partial fragments of Sequence ID No. 1 or derivatives thereof can
be generated and tested for their positive effect on productivity
as described in Examples 3 and 5. Some Examples of possible
fragments are shown in FIG. 12. The results obtained hitherto
indicate for example that the regions of Sequence ID No. 1 shown in
FIG. 12 could also bring about an increase in gene expression. In
stable transfection series these new TE elements are to be
characterised more closely with regard to their effect on specific
productivity in order to locate and further narrow down the
sequence regions which are important for the function. Narrowing
down of the function to specific sequence regions and the
associated possible reduction in the fragment length is
advantageous for efficient use in expression vectors, as smaller
expression plasmids are more stable and are easier to manipulate
both during cloning and during transfection.
[0301] Furthermore, it is possible to arrange similar or different
fragment regions in any orientation to one another and also in any
position within the plasmid. The investigation as to which of the
combinations results in the best possible increase in expression
can also be carried out in stable transfection series. Some
embodiments, which are in no way restrictive, are shown in FIG. 13.
Thus, for example, the investigation should determine whether the
TE elements TE-06 and TE-08 are able to bring about an additional
increase in expression when they flank the product gene on both
sides or are arranged one after another. Also, it is conceivable
that the concatination of short TE elements, be they identical or
different, such as TE element 06 and 08, for example, could also
lead to an additional expression-enhancing effect.
Further TE Elements
TABLE-US-00004 [0302] TE-ELEMENT 13 (SEQUENCE ID NO. 15)
gttgctattttagagacaggatttatgcaaacctggttggtcttaaactccgtatgtagctgagaatgaccttg-
aaaaccttcctgtccc
acccctcaaattccagaattatagacacccaccacatggcttaataagtaaacaacaacaataaaagcatgact-
tctgggtctggag
ggagggcttgccagttaagagcaatggatactttcccatagaacctgggtttgactcccagcactaacctacat-
ggtgatagtgatg
cagcagacatacatgagggcaacacacacatgggcacatacacacgcacccgcccaccatggatttcccccatc-
acttagacag
ccatatttaaacgtagtggagccaggctggggtggtggcccacacctttaatcccagcactccagaaggcagag-
gtaggcggatc
tctgtgggtttgagaccagcctggtctacaagagctagttccaggacagcctccaaagccatagagaaacccta-
tc TE-ELEMENT 14 (SEQUENCE ID NO. 16)
caaagccatagagaaaccctatctcaaaaaactgaaacaacaacaacaacaaaacaaaataaaaaaacaacaaa-
agaatcttagt
ggttcagtggttccacacacaggaaagtagaaagggccttgatgggaaggttttcagagggaggagtatggatg-
agacaggatg
atagtgaaaagaactcaaattaattaaatatttgaaactatctaagaataaaagctaaaatatttaaaattaca-
gtcaggtagtggtggt
gcagagggctaagttggtagacacagtgagatccaggccagccagggctacctagtgagaccttgttcaaataa-
ctaataaaatat
acaaaataaaggagacaccacaataattttgaaatgtaaaagactaaatttaccttttatattgatgagttgga-
taaaaaaatcaatttac
cagagaacataaagtagtcccatcaaagacaaaagcaatatatgattaaactctaatttaaaagtttgttagag-
cctggcaacgtggc acatacctttaatcccagcaccagg TE-ELEMENT 15 (SEQUENCE ID
NO. 17)
gttgctattttagagacaggatttatgcaaacctggttggtcttaaactccgtatgtagctgagaatgaccttg-
aaaaccttcctgtccc
acccctcaaattccagaattatagacacccaccacatggcttaataagtaaacaacaacaataaaagcatgact-
tctgggtctggag
ggagggcttgccagttaagagcaatggatactttcccatagaacctgggtttgactcccagcactaacctacat-
ggtgatagtgatg
cagcagacatacatgagggcaacacacacatgggcacatacacacgcacccgcccaccatggatttcccccatc-
acttagacag
ccatatttaaacgtagtggagccaggctggggtggtggcccacacctttaatcccagcactccagaaggcagag-
gtaggcggatc
tctgtgggtttgagaccagcctggtctacaagagctagttccaggacagcctccaaagccatagagaaacccta-
tctcaaaaaact
gaaacaacaacaacaacaaaacaaaataaaaaaacaacaaaagaatcttagtggttcagtggttccacacacag-
gaaagtagaaa
gggccttgatgggaaggttttcagagggaggagtatggatgagacaggatgatagtgaaaagaactcaaattaa-
ttaaatatttgaa
actatctaagaataaaagctaaaatatttaaaattacagtcaggtagtggtggtgcagagggctaagttggtag-
acacagtgagatc
caggccagccagggctacctagtgagaccttgttcaaataactaataaaatatacaaaataaaggagacaccac-
aataattttgaaa
tgtaaaagactaaatttaccttttatattgatgagttggataaaaaaatcaatttaccagagaacataaagtag-
tcccatcaaagacaaa
agcaatatatgattaaactctaatttaaaagtttgttagagcctggcaacgtggcacatacctttaatcccagc-
accagg TE-ELEMENT 16 (SEQUENCE ID NO. 18)
acctttaatcccagcaccagggagacagaggccatcctggtctaaaaagtgatctccaggacagccatggctat-
tacacagagaa
accctgtctggaaaaacaaaaaattagtgtccatgtgtaaatgtgtggagtatgcttgtcatgccacatacaga-
ggtagagggcagtt
tatgggagtcagttcctattatcctttatgggggacctggggactgaactcaggtcatcaggcttggcagaaag-
tgcattagctcac
ggagccttatcattggcgaaagctctctcaagtagaaaatcaatgtgtttgctcatagtgcaatcattatgttt-
cgagaggggaagggt
acaatcgttggggcatgtgtggtcacatctgaatagcagtagctccctaggagaattaattccaagttctttgg-
tggtgtatcaatgcc
cttaaaggggtcaacaactttttttccctctgacaaaactatcttcttatgtccttgtccc
TE-ELEMENT 17 (SEQUENCE ID NO. 19)
caaagccatagagaaaccctatctcaaaaaactgaaacaacaacaacaacaaaacaaaataaaaaaacaacaaa-
agaatcttagt
ggttcagtggttccacacacaggaaagtagaaagggccttgatgggaaggttttcagagggaggagtatggatg-
agacaggatg
atagtgaaaagaactcaaattaattaaatatttgaaactatctaagaataaaagctaaaatatttaaaattaca-
gtcaggtagtggtggt
gcagagggctaagttggtagacacagtgagatccaggccagccagggctacctagtgagaccttgttcaaataa-
ctaataaaatat
acaaaataaaggagacaccacaataattttgaaatgtaaaagactaaatttaccttttatattgatgagttgga-
taaaaaaatcaatttac
cagagaacataaagtagtcccatcaaagacaaaagcaatatatgattaaactctaatttaaaagtttgttagag-
cctggcaacgtggc
acatacctttaatcccagcaccagggagacagaggccatcctggtctaaaaagtgatctccaggacagccatgg-
ctattacacaga
gaaaccctgtctggaaaaacaaaaaattagtgtccatgtgtaaatgtgtggagtatgcttgtcatgccacatac-
agaggtagagggc
agtttatgggagtcagttcctattatcctttatgggggacctggggactgaactcaggtcatcaggcttggcag-
aaagtgcattagct
cacggagccttatcattggcgaaagctctctcaagtagaaaatcaatgtgtttgctcatagtgcaatcattatg-
tttcgagaggggaag
ggtacaatcgttggggcatgtgtggtcacatctgaatagcagtagctccctaggagaattaattccaagttctt-
tggtggtgtatcaat
gccataaaggggtcaacaactttttttccctctgacaaaactatcttcttatgtccttgtccc
TE-ELEMENT 18 (SEQUENCE ID NO. 20)
gttgctattttagagacaggatttcttgcaaacctggttggtcttaaactccgtatgtagctgagaatgacctt-
gaaaaccttcctgtccc
acccctcaaattccagaattatagacacccaccacatggcttaataagtaaacaacaacaataaaagcatgact-
tctgggtctggag
ggagggcttgccagttaagagcaatggatactttcccatagaacctgggtttgactcccagcactaacctacat-
ggtgatagtgatg
cagcagacatacatgagggcaacacacacatgggcacatacacacgcacccgcccaccatggcttttcccccat-
cacttagacag
ccatatttaaacgtagtggagccaggctggggtggtggcccacacctttaatcccagcactccagaaggcagag-
gtaggcggatc
tctgtgggtttgagaccagcctggtctacaagagctagttccaggacagcctccaaagccatagagaaacccta-
tctcaaaaaact
gaaacaacaacaacaacaaaacaaaataaaaaaacaacaaaagaatcttagtggttcagtggttccacacacag-
gaaagtagaaa
gggccttgatgggaaggttttcagagggaggagtatggatgagacaggatgatagtgaaaagaactcaaattaa-
ttaaatatttgaa
actatctaagaataaaagctaaaatatttaaaattacagtcaggtagtggtggtgcagagggctaagttggtag-
acacagtgagatc
caggccagccagggctacctagtgagaccttgttcaaataactaataaaatatacaaaataaaggagacaccac-
aataattttgaaa
tgtaaaagactaaatttaccttttatattgatgagttggataaaaaaatcaatttaccagagaacataaagtag-
tcccatcaaagacaaa
agcaatatatgattaaactctaatttaaaagtttgttagagcctggcaacgtggcacatacctttaatcccagc-
accagggagacaga
ggccatcctggtctaaaaagtgatctccaggacagccatggctattacacagagaaaccctgtctggaaaaaca-
aaaaattagtgt
ccatgtgtaaatgtgtggagtatgcttgtcatgccacatacagaggtagagggcagtttatgggagtcagttcc-
tattcttcctttatgg
gggacctggggactgaactcaggtcatcaggcttggcagaaagtgcattagctcacggagccttatcattggcg-
aaagctctctca
agtagaaaatcaatgtgtttgctcatagtgcaatcattatgtttcgagaggggaagggtacaatcgttggggca-
tgtgtggtcacatct
gaatagcagtagctccctaggagaattaattccaagttctttggtggtgtatcaatgcccttaaaggggtcaac-
aactttttttccctctg acaaaactatcttcttatgtccttgtccc TE-ELEMENT 21
(SEQUENCE ID NO. 21)
cttgcggtcgaggactacagtcattttgcaggtttccttactgtatggcttttaaaacgtgcaaaggtgaccat-
taaccgtttcacgctg
ggagggcacgtgcggctcagatgcttcctctgactgagggccaggagggggctacacggaagaggccacacccg-
cacttggg
aagactcgatttgggatcagctggctgagacgccccagcaggctcctcggctacaccttcagccccgaatgcct-
tccggcccata
acccttcccttctaggcatttccggcgaggacccaccctcgcgccaaacattcggccccatcccccggtcctca-
cctgaatctctaa ctctgactccagagtttagagactataaccagatag
Example 7
Influence of TE Elements RE-13 to TE-18 on the Expression of
MCP-1
[0303] The effect of the TE elements TE-13 to TE-18 on the
expression of the secreted MCP-1 was investigated in a stable
transfection series (Series F) of CHO-DG44 cells by comparison with
expression without the TE element. Four pools were produced per
plasmid variant. The base plasmid was pTE4/MCP-1 in all the series
(FIG. 1B; Selectable Marker NPT--Neomycin-phosphotransferase
F240I). The various plasmid variants contained either no TE element
(=control mixtures) or one of TE elements TE-13 to TE-18 in direct
orientation upstream from the promoter/enhancer (FIG. 12). In order
to minimise any influence on transfection efficiency caused by
different amounts of DNA in the transfection mixture, 1.2 .mu.g of
plasmid DNA were used in total in each case. Depending on the size
of the TE element introduced, the plasmid size varied between 6.7
kb and 8.2 kb. As a negative control, a mock-transfected pool was
run at the same time in each transfection series, i.e. treated the
same, but without the addition of DNA in the transfection mixture.
The selection of stably transfected cells took place two days after
transfection, using HT-supplemented CHO-S-SFMII+G418 (400
.mu.g/mL).
[0304] MCP-1 product titres and the specific productivity were
obtained over a period of 5 to 6 passages (passaging rhythm 2-2-3
days). FIG. 14 (Series F) shows the relative specific MCP-1
productivities. Each of the elements leads to an increase in the
average MCP-1 expression. The greatest increase (15-fold) was
obtained using element 13, which even exceeds the 10-fold increase
produced by element 08.
Example 8
Influence of the TE Elements at Various Positions and in Various
Combinations on the Expression of MCP-1
[0305] The effect of the TE elements TE-06 and TE-08 in various
combinations and at various positions in the expression plasmid on
the expression of the secreted MCP-1 was investigated in two stable
transfection series (Series G and H) of CHO-DG44 cells compared
with expression without the TE element. In both series, 6 pools
were produced per plasmid variant. The base plasmid was pTE4/MCP-1
(FIG. 1B; Selectable Marker NPT=Neomycin=phophotransferase F240I).
The different plasmid variants contained either no TE element
(=control mixtures) or TE-08 or TE-A in front of the
enhancer/promoter element or the combination of TE-0-6 and TE-08 in
front of the enhancer/promoter element or TE-08 or TE-09 in reverse
orientation in front of the enhancer/promoter element (Series G).
In Series H the elements TE-06 and TE-21 or TE-08 are used in front
of the enhancer/promoter element (E/P) and additionally after the
termination signal (T) (FIG. 13). In order to minimise any effect
on transfection efficiency caused by different amounts of DNA in
the transfection mixture, 1.2 .mu.g of plasmid DNA were used in
total in each case. Depending on the size of the TE element
introduced the plasmid size varied between 6.7 kb and 10.2 kb. As a
negative control, a mock-transfected pool was run at the same time
as each transfection series, i.e. treated the same but without the
addition of DNA in the transfection mixture. The selection of
stably transfected cells took place two days after transfection,
with HT-supplemented CHO-S-SFMII+G418 (300 .mu.g/mL).
[0306] The MCP-1 product titre and specific productivity in Series
G were obtained over a period of 6 passages (passaging rhythm 2-2-3
days). The same procedure is used in Series H as well. FIG. 15
shows the relative specific MCP-1 productivities of Series G. All
the elements lead to an increase in the average MCP-1 expression.
The greatest increase (4-fold) was produced by the element TE-A.
The use of the elements TE-06 and TE-21 or TE-08 before and after
the expression cassette gave rise to an increase.
Example 9
Influence of TE Element TE-08 on the Expression of Two
Immunoglobulins G-4(IgG-4)
[0307] The effect of TE element TE-08 on the expression of two
IgG-4 antibodies is investigated in a stable transfection series
(Series J) of CHO-DG44 cells by comparison with the expression
without the TE element. 24 pools are produced with the base
plasmids pBIN-LC2 or. pBIN-LC3 and pBID-HC2 or. pBID-HC3 and 24
pools are produced with pBIN-LC2/TE08 or. pBIN-LC3/TE08 and
pBID-HC2/TE08 or. pBID-HC3/TE08 (FIG. 16; Selectable Marker
NPT=Neomycin-phosphotransferase F240I and
dhfr=Dihydrofolate-reductase). In order to minimise any influence
on transfection efficiency caused by varying amounts of DNA in the
transfection mixture, 1.2 .mu.g of plasmid DNA are used in total in
each case. Depending on the size of the TE element introduced, the
plasmid size varies between 6.1 kb and 7.5 kb. As a negative
control, a mock-transfected pool is run at the same time with each
transfection series, i.e. treated the same, but without the
addition of DNA to the transfection mixture. The selection of
stably transfected cells is carried out two days after
transfection, using HT-free CHO-S-SFMII+G418 (400 .mu.g/mL).
[0308] IgG-4 product titres and the specific productivity are
obtained over a period of 4 passages (passaging rhythm 2-2-3 days).
The element 08 leads to an increase in the average expression rate
in the expression of IgG4 antibodies. Moreover, the chance of
finding a high producing cell pool can be increased by the presence
of the element TE-08.
Example 10
Influence of TE Elements on Protein Expression in 293F Cells
[0309] The effect of various TE elements on the expression of the
secreted MCP-1 is investigated in a stable transfection series
(Series K) of HEK293 freestyle cells by comparison with MCP-1
expression without a TE element. The base plasmid is pTE-4/MCP-1
(FIG. 1B; Selectable Marker NPT=Neomycin-phosphotransferase F240I).
The elements TE-08, TE-13 and TE-A are used in direct orientation
upstream from the enhancer/promoter and 7-10 pools are produced per
plasmid variant. In order to minimise any effect on transfection
efficiency caused by different amounts of DNA in the transfection
mixture, 1.2 .mu.g of plasmid DNA are used in total in each case.
Depending on the size of the TE element introduced the plasmid size
varies between 6.7 kb and 10.2 kb. As a negative control a
mock-transfected pool is run at the same time as each transfection
series, i.e. treated the same but without the addition of DNA to
the transfection mixture. The selection of stably transfected cells
takes place two days after the transfection with 293 SFM II medium
+4 mM glutamin+G418 (100 .mu.g/ml).
[0310] MCP-1 product titre and the specific productivity are
obtained over a period of 5 to 6 passages (passaging rhythm 2-2-3
days).
Example 11
Influence of the TE Element TE-08 on the Expression of an Enzyme
(SEAP)
[0311] The effect of the TE element TE-08 on the expression of an
enzyme (SEAP) is investigated in a stable transfection series
(Series L) of CHO-DG44 cells compared with SEAP expression without
the TE element. Six pools are produced per plasmid variant. The
base plasmid is pTE-4/SEAP. It is generated by exchanging
MCP-1--IRES--DsRed2-expression cassette for SEAP. The element TE-08
is cloned into the adaptor A (FIG. 1B; Selectable Marker
NPT=Neomycin-phosphotransferase F240I). In order to minimise any
effect on the transfection efficiency caused by varying amounts of
DNA in the transfection mixture, 1.2 .mu.g of plasmid DNA are used
in total in each case. Depending on the size of the TE element
introduced the plasmid size varies between 6.6 kb and 7.6 kb. As a
negative control a mock-transfected pool is run at the same time as
each transfection series, i.e. treated the same but without the
addition of DNA to the transfection mixture. The selection of
stably transfected cells takes place two days after transfection,
with HT-supplemented CHO-S-SFMII+G418 (400 .mu.g/mL).
[0312] The relative SEAP expression is determined using the
commercially obtainable SEAP assay (Clontech) and obtained over a
period of 6 passages (passaging rhythm 2-2-3 days).
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EP-0-393-438 [0371] WO2004/050884 [0372] WO02/081677 [0373] U.S.
Pat. No. 6,027,915 [0374] U.S. Pat. No. 6,309,851 [0375] WO01/04306
[0376] WO00/34318 [0377] WO00/34326 [0378] WO00/34526 [0379]
WO01/27150 [0380] U.S. Pat. No. 5,122,458 [0381] WO94/05785 [0382]
WO92/08796 [0383] WO94/28143 [0384] WO03/004704
[0385] What is claimed is:
Sequence CWU 1
1
4413788DNAArtificialCricetulus griseus derivative, additional 8
nucleotides 1ccatgagagc ctgaagacct gagttgatac ccagaaccca gatcaagatg
gaggagagaa 60ccagccccac taagctgtcc cctgaccccc ataaatgcct ccctgtccag
ttatgccaca 120caatgatagg tgaatacaga aaaacaccct tcctttagac
actaagcgga ttcctcttac 180gcataccagt taagtgatag ttcttaggct
tcaactcagc actttaaaaa gtttatattt 240tgcaatgctg gggactaaat
tagggttgtg cacatgctaa gtaagcactc tacttttgta 300tcacatttta
ataattgtaa gaattaattc gtgaaatagt agctgagaca atagatttgt
360ttctttcatg tgggaactgc tgtgtgtgct tcttgctgat gcaaacaagg
tcaaatactt 420tattccccag tgtctgccta gccctgtaac acttctctat
tatacaatga ccacaaataa 480ttaggtgagt gggttttgtt tcattttaaa
ttgttgctat tttagagaca ggatttcttg 540caaacctggt tggtcttaaa
ctccgtatgt agctgagaat gaccttgaaa accttcctgt 600cccacccctc
aaattccaga attatagaca cccaccacat ggcttaataa gtaaacaaca
660acaataaaag catgacttct gggtctggag ggagggcttg ccagttaaga
gcaatggata 720ctttcccata gaacctgggt ttgactccca gcactaacct
acatggtgat agtgatgcag 780cagacataca tgagggcaac acacacatgg
gcacatacac acgcacccgc ccaccatggc 840ttttccccca tcacttagac
agccatattt aaacgtagtg gagccaggct ggggtggtgg 900cccacacctt
taatcccagc actccagaag gcagaggtag gcggatctct gtgggtttga
960gaccagcctg gtctacaaga gctagttcca ggacagcctc caaagccata
gagaaaccct 1020atctcaaaaa actgaaacaa caacaacaac aaaacaaaat
aaaaaaacaa caaaagaatc 1080ttagtggttc agtggttcca cacacaggaa
agtagaaagg gccttgatgg gaaggttttc 1140agagggagga gtatggatga
gacaggatga tagtgaaaag aactcaaatt aattaaatat 1200ttgaaactat
ctaagaataa aagctaaaat atttaaaatt acagtcaggt agtggtggtg
1260cagagggcta agttggtaga cacagtgaga tccaggccag ccagggctac
ctagtgagac 1320cttgttcaaa taactaataa aatatacaaa ataaaggaga
caccacaata attttgaaat 1380gtaaaagact aaatttacct tttatattga
tgagttggat aaaaaaatca atttaccaga 1440gaacataaag tagtcccatc
aaagacaaaa gcaatatatg attaaactct aatttaaaag 1500tttgttagag
cctggcaacg tggcacatac ctttaatccc agcaccaggg agacagaggc
1560catcctggtc taaaaagtga tctccaggac agccatggct attacacaga
gaaaccctgt 1620ctggaaaaac aaaaaattag tgtccatgtg taaatgtgtg
gagtatgctt gtcatgccac 1680atacagaggt agagggcagt ttatgggagt
cagttcctat tcttccttta tgggggacct 1740ggggactgaa ctcaggtcat
caggcttggc agaaagtgca ttagctcacg gagccttatc 1800attggcgaaa
gctctctcaa gtagaaaatc aatgtgtttg ctcatagtgc aatcattatg
1860tttcgagagg ggaagggtac aatcgttggg gcatgtgtgg tcacatctga
atagcagtag 1920ctccctagga gaattaattc caagttcttt ggtggtgtat
caatgccctt aaaggggtca 1980acaacttttt ttccctctga caaaactatc
ttcttatgtc cttgtccctc atatttgaag 2040tattttattc tttgcagtgt
tgaatatcaa ttctagcacc tcagacatgt taggtaagta 2100ccctacaact
caggttaact aatttaattt aactaattta accccaacac tttttctttg
2160tttatccaca tttgtggagt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
gtgtgtgtgt 2220gtgtgtgtgt gtgtgtgtgt gcgcgcgcgc gcgcgctcgg
atcattctac cttttgttta 2280aaaaatgtta gtccaggggt ggggtgcact
gtgaaagtct gagggtaact tgctggggtc 2340agttctttcc actataggac
agaactccag gtgtcaactc tttactgaca gaaccatcca 2400aatagcccta
tctaatttta gttttttatt tatttatttt ttgtttttcg agacagggtt
2460tctctgtggc tttggaggct gtcctggaac tagctcttgt agaccaggct
ggtctcgaac 2520tcagagatcc acctgcctct gcctcctgag tgctgggatt
aaaggcatgc gccaccaacg 2580cttggctcta cctaatttta aaagagattg
tgtgtcacaa gggtgtcatg tcgccctgca 2640accacccccc ccccaaaaaa
aaaaaaaaaa aaacttcact gaagctgaag cacgatgatt 2700tggttactct
ggctggccaa tgagctctag ggagtctcct gtcaaacaga atctcaacag
2760gcgcagcagt cttttttaaa gtggggttac aacacaggtt tttgcatatc
aggcatttta 2820tctaagctat ttcccagcca aaaatgtgta ttttggaggc
agcagagcta atagattaaa 2880atgagggaag agcccacaca ggttattagg
aagataagca tcttctttat ataaaacaaa 2940accaaaccaa actggaggag
gtctaccttt agggatggaa gaaaagacat ttagagggtg 3000caatagaaag
ggcactgagt ttgtgaggtg gaggactggg agagggcgca accgctttaa
3060ctgtcctgtt ttgcctattt tttggggaca gcacatgttc ctatttttcc
caggatgggc 3120aatctccacg tccaaacttg cggtcgagga ctacagtcat
tttgcaggtt tccttactgt 3180atggctttta aaacgtgcaa aggtgaccat
taaccgtttc acgctgggag ggcacgtgcg 3240gctcagatgc ttcctctgac
tgagggccag gagggggcta cacggaagag gccacacccg 3300cacttgggaa
gactcgattt gggcttcagc tggctgagac gccccagcag gctcctcggc
3360tacaccttca gccccgaatg ccttccggcc cataaccctt cccttctagg
catttccggc 3420gaggacccac cctcgcgcca aacattcggc cccatccccc
ggtcctcacc tgaatctcta 3480actctgactc cagagtttag agactataac
cagatagccc ggatgtgtgg aactgcatct 3540tgggacgagt agttttagca
aaaagaaagc gacgaaaaac tacaattccc agacagactt 3600gtgttacctc
tcttctcatg ctaaacaagc cccctttaaa ggaaagcccc tcttagtcgc
3660atcgactgtg taagaaaggc gtttgaaaca ttttaatgtt gggcacaccg
tttcgaggac 3720cgaaatgaga aagagcatag ggaaacggag cgcccgagct
agtctggcac tgcgttagac 3780agccgcgg 378822210DNACricetulus griseus
2gatctccagg acagccatgg ctattacaca gagaaaccct gtctggaaaa acaaaaaatt
60agtgtccatg tgtaaatgtg tggagtatgc ttgtcatgcc acatacagag gtagagggca
120gtttatggga gtcagttcct attcttcctt tatgggggac ctggggactg
aactcaggtc 180atcaggcttg gcagaaagtg cattagctca cggagcctta
tcattggcga aagctctctc 240aagtagaaaa tcaatgtgtt tgctcatagt
gcaatcatta tgtttcgaga ggggaagggt 300acaatcgttg gggcatgtgt
ggtcacatct gaatagcagt agctccctag gagaattaat 360tccaagttct
ttggtggtgt atcaatgccc ttaaaggggt caacaacttt ttttccctct
420gacaaaacta tcttcttatg tccttgtccc tcatatttga agtattttat
tctttgcagt 480gttgaatatc aattctagca cctcagacat gttaggtaag
taccctacaa ctcaggttaa 540ctaatttaat ttaactaatt taaccccaac
actttttctt tgtttatcca catttgtgga 600gtgtgtgtgt gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 660gtgcgcgcgc
gcgcgcgctc ggatcattct accttttgtt taaaaaatgt tagtccaggg
720gtggggtgca ctgtgaaagt ctgagggtaa cttgctgggg tcagttcttt
ccactatagg 780acagaactcc aggtgtcaac tctttactga cagaaccatc
caaatagccc tatctaattt 840tagtttttta tttatttatt ttttgttttt
cgagacaggg tttctctgtg gctttggagg 900ctgtcctgga actagctctt
gtagaccagg ctggtctcga actcagagat ccacctgcct 960ctgcctcctg
agtgctggga ttaaaggcat gcgccaccaa cgcttggctc tacctaattt
1020taaaagagat tgtgtgtcac aagggtgtca tgtcgccctg caaccacccc
ccccccaaaa 1080aaaaaaaaaa aaaaacttca ctgaagctga agcacgatga
tttggttact ctggctggcc 1140aatgagctct agggagtctc ctgtcaaaca
gaatctcaac aggcgcagca gtctttttta 1200aagtggggtt acaacacagg
tttttgcata tcaggcattt tatctaagct atttcccagc 1260caaaaatgtg
tattttggag gcagcagagc taatagatta aaatgaggga agagcccaca
1320caggttatta ggaagataag catcttcttt atataaaaca aaaccaaacc
aaactggagg 1380aggtctacct ttagggatgg aagaaaagac atttagaggg
tgcaatagaa agggcactga 1440gtttgtgagg tggaggactg ggagagggcg
caaccgcttt aactgtcctg ttttgcctat 1500tttttgggga cagcacatgt
tcctattttt cccaggatgg gcaatctcca cgtccaaact 1560tgcggtcgag
gactacagtc attttgcagg tttccttact gtatggcttt taaaacgtgc
1620aaaggtgacc attaaccgtt tcacgctggg agggcacgtg cggctcagat
gcttcctctg 1680actgagggcc aggagggggc tacacggaag aggccacacc
cgcacttggg aagactcgat 1740ttgggcttca gctggctgag acgccccagc
aggctcctcg gctacacctt cagccccgaa 1800tgccttccgg cccataaccc
ttcccttcta ggcatttccg gcgaggaccc accctcgcgc 1860caaacattcg
gccccatccc ccggtcctca cctgaatctc taactctgac tccagagttt
1920agagactata accagatagc ccggatgtgt ggaactgcat cttgggacga
gtagttttag 1980caaaaagaaa gcgacgaaaa actacaattc ccagacagac
ttgtgttacc tctcttctca 2040tgctaaacaa gcccccttta aaggaaagcc
cctcttagtc gcatcgactg tgtaagaaag 2100gcgtttgaaa cattttaatg
ttgggcacac cgtttcgagg accgaaatga gaaagagcat 2160agggaaacgg
agcgcccgag ctagtctggc actgcgttag acagccgcgg
221033005DNAArtificialCricetulus griseus with manipulation of the
endogenous EcoR1 site by substitution of 4 bases 3gttgctattt
tagagacagg atttcttgca aacctggttg gtcttaaact ccgtatgtag 60ctgagaatga
ccttgaaaac cttcctgtcc cacccctcaa attccagaat tatagacacc
120caccacatgg cttaataagt aaacaacaac aataaaagca tgacttctgg
gtctggaggg 180agggcttgcc agttaagagc aatggatact ttcccataga
acctgggttt gactcccagc 240actaacctac atggtgatag tgatgcagca
gacatacatg agggcaacac acacatgggc 300acatacacac gcacccgccc
accatggctt ttcccccatc acttagacag ccatatttaa 360acgtagtgga
gccaggctgg ggtggtggcc cacaccttta atcccagcac tccagaaggc
420agaggtaggc ggatctctgt gggtttgaga ccagcctggt ctacaagagc
tagttccagg 480acagcctcca aagccataga gaaaccctat ctcaaaaaac
tgaaacaaca acaacaacaa 540aacaaaataa aaaaacaaca aaagaatctt
agtggttcag tggttccaca cacaggaaag 600tagaaagggc cttgatggga
aggttttcag agggaggagt atggatgaga caggatgata 660gtgaaaagaa
ctcaaattaa ttaaatattt gaaactatct aagaataaaa gctaaaatat
720ttaaaattac agtcaggtag tggtggtgca gagggctaag ttggtagaca
cagtgagatc 780caggccagcc agggctacct agtgagacct tgttcaaata
actaataaaa tatacaaaat 840aaaggagaca ccacaataat tttgaaatgt
aaaagactaa atttaccttt tatattgatg 900agttggataa aaaaatcaat
ttaccagaga acataaagta gtcccatcaa agacaaaagc 960aatatatgat
taaactctaa tttaaaagtt tgttagagcc tggcaacgtg gcacatacct
1020ttaatcccag caccagggag acagaggcca tcctggtcta aaaagtgatc
tccaggacag 1080ccatggctat tacacagaga aaccctgtct ggaaaaacaa
aaaattagtg tccatgtgta 1140aatgtgtgga gtatgcttgt catgccacat
acagaggtag agggcagttt atgggagtca 1200gttcctattc ttcctttatg
ggggacctgg ggactgaact caggtcatca ggcttggcag 1260aaagtgcatt
agctcacgga gccttatcat tggcgaaagc tctctcaagt agaaaatcaa
1320tgtgtttgct catagtgcaa tcattatgtt tcgagagggg aagggtacaa
tcgttggggc 1380atgtgtggtc acatctgaat agcagtagct ccctaggaga
attaattcca agttctttgg 1440tggtgtatca atgcccttaa aggggtcaac
aacttttttt ccctctgaca aaactatctt 1500cttatgtcct tgtccctcat
atttgaagta ttttattctt tgcagtgttg aatatcaatt 1560ctagcacctc
agacatgtta ggtaagtacc ctacaactca ggttaactaa tttaatttaa
1620ctaatttaac cccaacactt tttctttgtt tatccacatt tgtggagtgt
gtgtgtgtgt 1680gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
gtgtgtgtgc gcgcgcgcgc 1740gcgctcggat cattctacct tttgtttaaa
aaatgttagt ccaggggtgg ggtgcactgt 1800gaaagtctga gggtaacttg
ctggggtcag ttctttccac tataggacag aactccaggt 1860gtcaactctt
tactgacaga accatccaaa tagccctatc taattttagt tttttattta
1920tttatttttt gtttttcgag acagggtttc tctgtggctt tggaggctgt
cctggaacta 1980gctcttgtag accaggctgg tctcgaactc agagatccac
ctgcctctgc ctcctgagtg 2040ctgggattaa aggcatgcgc caccaacgct
tggctctacc taattttaaa agagattgtg 2100tgtcacaagg gtgtcatgtc
gccctgcaac cacccccccc ccaaaaaaaa aaaaaaaaaa 2160acttcactga
agctgaagca cgatgatttg gttactctgg ctggccaatg agctctaggg
2220agtctcctgt caaacagaat ctcaacaggc gcagcagtct tttttaaagt
ggggttacaa 2280cacaggtttt tgcatatcag gcattttatc taagctattt
cccagccaaa aatgtgtatt 2340ttggaggcag cagagctaat agattaaaat
gagggaagag cccacacagg ttattaggaa 2400gataagcatc ttctttatat
aaaacaaaac caaaccaaac tggaggaggt ctacctttag 2460ggatggaaga
aaagacattt agagggtgca atagaaaggg cactgagttt gtgaggtgga
2520ggactgggag agggcgcaac cgctttaact gtcctgtttt gcctattttt
tggggacagc 2580acatgttcct atttttccca ggatgggcaa tctccacgtc
caaacttgcg gtcgaggact 2640acagtcattt tgcaggtttc cttactgtat
ggcttttaaa acgtgcaaag gtgaccatta 2700accgtttcac gctgggaggg
cacgtgcggc tcagatgctt cctctgactg agggccagga 2760gggggctaca
cggaagaggc cacacccgca cttgggaaga ctcgatttgg gcttcagctg
2820gctgagacgc cccagcaggc tcctcggcta caccttcagc cccgaatgcc
ttccggccca 2880taacccttcc cttctaggca tttccggcga ggacccaccc
tcgcgccaaa cattcggccc 2940catcccccgg tcctcacctg aatctctaac
tctgactcca gagtttagag actataacca 3000gatag 300542517DNACricetulus
griseus 4caaagccata gagaaaccct atctcaaaaa actgaaacaa caacaacaac
aaaacaaaat 60aaaaaaacaa caaaagaatc ttagtggttc agtggttcca cacacaggaa
agtagaaagg 120gccttgatgg gaaggttttc agagggagga gtatggatga
gacaggatga tagtgaaaag 180aactcaaatt aattaaatat ttgaaactat
ctaagaataa aagctaaaat atttaaaatt 240acagtcaggt agtggtggtg
cagagggcta agttggtaga cacagtgaga tccaggccag 300ccagggctac
ctagtgagac cttgttcaaa taactaataa aatatacaaa ataaaggaga
360caccacaata attttgaaat gtaaaagact aaatttacct tttatattga
tgagttggat 420aaaaaaatca atttaccaga gaacataaag tagtcccatc
aaagacaaaa gcaatatatg 480attaaactct aatttaaaag tttgttagag
cctggcaacg tggcacatac ctttaatccc 540agcaccaggg agacagaggc
catcctggtc taaaaagtga tctccaggac agccatggct 600attacacaga
gaaaccctgt ctggaaaaac aaaaaattag tgtccatgtg taaatgtgtg
660gagtatgctt gtcatgccac atacagaggt agagggcagt ttatgggagt
cagttcctat 720tcttccttta tgggggacct ggggactgaa ctcaggtcat
caggcttggc agaaagtgca 780ttagctcacg gagccttatc attggcgaaa
gctctctcaa gtagaaaatc aatgtgtttg 840ctcatagtgc aatcattatg
tttcgagagg ggaagggtac aatcgttggg gcatgtgtgg 900tcacatctga
atagcagtag ctccctagga gaattaattc caagttcttt ggtggtgtat
960caatgccctt aaaggggtca acaacttttt ttccctctga caaaactatc
ttcttatgtc 1020cttgtccctc atatttgaag tattttattc tttgcagtgt
tgaatatcaa ttctagcacc 1080tcagacatgt taggtaagta ccctacaact
caggttaact aatttaattt aactaattta 1140accccaacac tttttctttg
tttatccaca tttgtggagt gtgtgtgtgt gtgtgtgtgt 1200gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gcgcgcgcgc gcgcgctcgg
1260atcattctac cttttgttta aaaaatgtta gtccaggggt ggggtgcact
gtgaaagtct 1320gagggtaact tgctggggtc agttctttcc actataggac
agaactccag gtgtcaactc 1380tttactgaca gaaccatcca aatagcccta
tctaatttta gttttttatt tatttatttt 1440ttgtttttcg agacagggtt
tctctgtggc tttggaggct gtcctggaac tagctcttgt 1500agaccaggct
ggtctcgaac tcagagatcc acctgcctct gcctcctgag tgctgggatt
1560aaaggcatgc gccaccaacg cttggctcta cctaatttta aaagagattg
tgtgtcacaa 1620gggtgtcatg tcgccctgca accacccccc ccccaaaaaa
aaaaaaaaaa aaacttcact 1680gaagctgaag cacgatgatt tggttactct
ggctggccaa tgagctctag ggagtctcct 1740gtcaaacaga atctcaacag
gcgcagcagt cttttttaaa gtggggttac aacacaggtt 1800tttgcatatc
aggcatttta tctaagctat ttcccagcca aaaatgtgta ttttggaggc
1860agcagagcta atagattaaa atgagggaag agcccacaca ggttattagg
aagataagca 1920tcttctttat ataaaacaaa accaaaccaa actggaggag
gtctaccttt agggatggaa 1980gaaaagacat ttagagggtg caatagaaag
ggcactgagt ttgtgaggtg gaggactggg 2040agagggcgca accgctttaa
ctgtcctgtt ttgcctattt tttggggaca gcacatgttc 2100ctatttttcc
caggatgggc aatctccacg tccaaacttg cggtcgagga ctacagtcat
2160tttgcaggtt tccttactgt atggctttta aaacgtgcaa aggtgaccat
taaccgtttc 2220acgctgggag ggcacgtgcg gctcagatgc ttcctctgac
tgagggccag gagggggcta 2280cacggaagag gccacacccg cacttgggaa
gactcgattt gggcttcagc tggctgagac 2340gccccagcag gctcctcggc
tacaccttca gccccgaatg ccttccggcc cataaccctt 2400cccttctagg
catttccggc gaggacccac cctcgcgcca aacattcggc cccatccccc
2460ggtcctcacc tgaatctcta actctgactc cagagtttag agactataac cagatag
251751989DNACricetulus griseus 5acctttaatc ccagcaccag ggagacagag
gccatcctgg tctaaaaagt gatctccagg 60acagccatgg ctattacaca gagaaaccct
gtctggaaaa acaaaaaatt agtgtccatg 120tgtaaatgtg tggagtatgc
ttgtcatgcc acatacagag gtagagggca gtttatggga 180gtcagttcct
attcttcctt tatgggggac ctggggactg aactcaggtc atcaggcttg
240gcagaaagtg cattagctca cggagcctta tcattggcga aagctctctc
aagtagaaaa 300tcaatgtgtt tgctcatagt gcaatcatta tgtttcgaga
ggggaagggt acaatcgttg 360gggcatgtgt ggtcacatct gaatagcagt
agctccctag gagaattaat tccaagttct 420ttggtggtgt atcaatgccc
ttaaaggggt caacaacttt ttttccctct gacaaaacta 480tcttcttatg
tccttgtccc tcatatttga agtattttat tctttgcagt gttgaatatc
540aattctagca cctcagacat gttaggtaag taccctacaa ctcaggttaa
ctaatttaat 600ttaactaatt taaccccaac actttttctt tgtttatcca
catttgtgga gtgtgtgtgt 660gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgcgcgcgc 720gcgcgcgctc ggatcattct
accttttgtt taaaaaatgt tagtccaggg gtggggtgca 780ctgtgaaagt
ctgagggtaa cttgctgggg tcagttcttt ccactatagg acagaactcc
840aggtgtcaac tctttactga cagaaccatc caaatagccc tatctaattt
tagtttttta 900tttatttatt ttttgttttt cgagacaggg tttctctgtg
gctttggagg ctgtcctgga 960actagctctt gtagaccagg ctggtctcga
actcagagat ccacctgcct ctgcctcctg 1020agtgctggga ttaaaggcat
gcgccaccaa cgcttggctc tacctaattt taaaagagat 1080tgtgtgtcac
aagggtgtca tgtcgccctg caaccacccc ccccccaaaa aaaaaaaaaa
1140aaaaacttca ctgaagctga agcacgatga tttggttact ctggctggcc
aatgagctct 1200agggagtctc ctgtcaaaca gaatctcaac aggcgcagca
gtctttttta aagtggggtt 1260acaacacagg tttttgcata tcaggcattt
tatctaagct atttcccagc caaaaatgtg 1320tattttggag gcagcagagc
taatagatta aaatgaggga agagcccaca caggttatta 1380ggaagataag
catcttcttt atataaaaca aaaccaaacc aaactggagg aggtctacct
1440ttagggatgg aagaaaagac atttagaggg tgcaatagaa agggcactga
gtttgtgagg 1500tggaggactg ggagagggcg caaccgcttt aactgtcctg
ttttgcctat tttttgggga 1560cagcacatgt tcctattttt cccaggatgg
gcaatctcca cgtccaaact tgcggtcgag 1620gactacagtc attttgcagg
tttccttact gtatggcttt taaaacgtgc aaaggtgacc 1680attaaccgtt
tcacgctggg agggcacgtg cggctcagat gcttcctctg actgagggcc
1740aggagggggc tacacggaag aggccacacc cgcacttggg aagactcgat
ttgggcttca 1800gctggctgag acgccccagc aggctcctcg gctacacctt
cagccccgaa tgccttccgg 1860cccataaccc ttcccttcta ggcatttccg
gcgaggaccc accctcgcgc caaacattcg 1920gccccatccc ccggtcctca
cctgaatctc taactctgac tccagagttt agagactata 1980accagatag
198961512DNACricetulus griseus 6ctatcttctt atgtccttgt ccctcatatt
tgaagtattt tattctttgc agtgttgaat 60atcaattcta gcacctcaga catgttaggt
aagtacccta caactcaggt taactaattt 120aatttaacta atttaacccc
aacacttttt ctttgtttat ccacatttgt ggagtgtgtg 180tgtgtgtgtg
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgcgcg
240cgcgcgcgcg ctcggatcat tctacctttt gtttaaaaaa tgttagtcca
ggggtggggt 300gcactgtgaa agtctgaggg taacttgctg gggtcagttc
tttccactat aggacagaac 360tccaggtgtc aactctttac tgacagaacc
atccaaatag ccctatctaa ttttagtttt 420ttatttattt attttttgtt
tttcgagaca gggtttctct gtggctttgg aggctgtcct 480ggaactagct
cttgtagacc aggctggtct cgaactcaga gatccacctg cctctgcctc
540ctgagtgctg ggattaaagg catgcgccac caacgcttgg ctctacctaa
ttttaaaaga 600gattgtgtgt cacaagggtg tcatgtcgcc ctgcaaccac
ccccccccca aaaaaaaaaa 660aaaaaaaact tcactgaagc tgaagcacga
tgatttggtt actctggctg gccaatgagc 720tctagggagt ctcctgtcaa
acagaatctc aacaggcgca gcagtctttt ttaaagtggg 780gttacaacac
aggtttttgc atatcaggca ttttatctaa gctatttccc agccaaaaat
840gtgtattttg gaggcagcag agctaataga ttaaaatgag ggaagagccc
acacaggtta 900ttaggaagat aagcatcttc tttatataaa acaaaaccaa
accaaactgg aggaggtcta 960cctttaggga tggaagaaaa gacatttaga
gggtgcaata gaaagggcac tgagtttgtg 1020aggtggagga ctgggagagg
gcgcaaccgc tttaactgtc ctgttttgcc
tattttttgg 1080ggacagcaca tgttcctatt tttcccagga tgggcaatct
ccacgtccaa acttgcggtc 1140gaggactaca gtcattttgc aggtttcctt
actgtatggc ttttaaaacg tgcaaaggtg 1200accattaacc gtttcacgct
gggagggcac gtgcggctca gatgcttcct ctgactgagg 1260gccaggaggg
ggctacacgg aagaggccac acccgcactt gggaagactc gatttgggct
1320tcagctggct gagacgcccc agcaggctcc tcggctacac cttcagcccc
gaatgccttc 1380cggcccataa cccttccctt ctaggcattt ccggcgagga
cccaccctcg cgccaaacat 1440tcggccccat cccccggtcc tcacctgaat
ctctaactct gactccagag tttagagact 1500ataaccagat ag
151271013DNACricetulus griseus 7caggctggtc tcgaactcag agatccacct
gcctctgcct cctgagtgct gggattaaag 60gcatgcgcca ccaacgcttg gctctaccta
attttaaaag agattgtgtg tcacaagggt 120gtcatgtcgc cctgcaacca
cccccccccc aaaaaaaaaa aaaaaaaaac ttcactgaag 180ctgaagcacg
atgatttggt tactctggct ggccaatgag ctctagggag tctcctgtca
240aacagaatct caacaggcgc agcagtcttt tttaaagtgg ggttacaaca
caggtttttg 300catatcaggc attttatcta agctatttcc cagccaaaaa
tgtgtatttt ggaggcagca 360gagctaatag attaaaatga gggaagagcc
cacacaggtt attaggaaga taagcatctt 420ctttatataa aacaaaacca
aaccaaactg gaggaggtct acctttaggg atggaagaaa 480agacatttag
agggtgcaat agaaagggca ctgagtttgt gaggtggagg actgggagag
540ggcgcaaccg ctttaactgt cctgttttgc ctattttttg gggacagcac
atgttcctat 600ttttcccagg atgggcaatc tccacgtcca aacttgcggt
cgaggactac agtcattttg 660caggtttcct tactgtatgg cttttaaaac
gtgcaaaggt gaccattaac cgtttcacgc 720tgggagggca cgtgcggctc
agatgcttcc tctgactgag ggccaggagg gggctacacg 780gaagaggcca
cacccgcact tgggaagact cgatttgggc ttcagctggc tgagacgccc
840cagcaggctc ctcggctaca ccttcagccc cgaatgcctt ccggcccata
acccttccct 900tctaggcatt tccggcgagg acccaccctc gcgccaaaca
ttcggcccca tcccccggtc 960ctcacctgaa tctctaactc tgactccaga
gtttagagac tataaccaga tag 10138381DNAArtificialmutant /point
mutation in a Cricetulus griseus sequence 8cttgcggtcg aggactacag
tcattttgca ggtttcctta ctgtatggct tttaaaacgt 60gcaaaggtga ccattaaccg
tttcacgctg ggagggcacg tgcggctcag atgcttcctc 120tgactgaggg
ccaggagggg gctacacgga agaggccaca cccgcacttg ggaagactcg
180atttgggctt cagctggctg agacgcccca gcaggctcct cggctacacc
ttcagccccg 240aatgccttcc ggcccataac ccttcccttc taggcatttc
cggcgaggac ccaccctcgc 300gccaaacatt cggccccatc ccccggtcct
cacctgaatc tctaactctg actccagagt 360ttagcgacta taaccagata g
3819529DNACricetulus griseus 9gcctgaagac ctgagttgat acccagaacc
cagatcaaga tggaggagag aaccagcccc 60actaagctgt cccctgaccc ccataaatgc
ctccctgtcc agttatgcca cacaatgata 120ggtgaataca gaaaaacacc
cttcctttag acactaagcg gattcctctt acgcatacca 180gttaagtgat
agttcttagg cttcaactca gcactttaaa aagtttatat tttgcaatgc
240tggggactaa attagggttg tgcacatgct aagtaagcac tctacttttg
tatcacattt 300taataattgt aagaattaat tcgtgaaata gtagctgaga
caatagattt gtttctttca 360tgtgggaact gctgtgtgtg cttcttgctg
atgcaaacaa ggtcaaatac tttattcccc 420agtgtctgcc tagccctgta
acacttctct attatacaat gaccacaaat aattaggtga 480gtgggttttg
tttcatttta aattgttgct attttagaga caggatttc 529101015DNACricetulus
griseus 10gcctgaagac ctgagttgat acccagaacc cagatcaaga tggaggagag
aaccagcccc 60actaagctgt cccctgaccc ccataaatgc ctccctgtcc agttatgcca
cacaatgata 120ggtgaataca gaaaaacacc cttcctttag acactaagcg
gattcctctt acgcatacca 180gttaagtgat agttcttagg cttcaactca
gcactttaaa aagtttatat tttgcaatgc 240tggggactaa attagggttg
tgcacatgct aagtaagcac tctacttttg tatcacattt 300taataattgt
aagaattaat tcgtgaaata gtagctgaga caatagattt gtttctttca
360tgtgggaact gctgtgtgtg cttcttgctg atgcaaacaa ggtcaaatac
tttattcccc 420agtgtctgcc tagccctgta acacttctct attatacaat
gaccacaaat aattaggtga 480gtgggttttg tttcatttta aattgttgct
attttagaga caggatttct tgcaaacctg 540gttggtctta aactccgtat
gtagctgaga atgaccttga aaaccttcct gtcccacccc 600tcaaattcca
gaattataga cacccaccac atggcttaat aagtaaacaa caacaataaa
660agcatgactt ctgggtctgg agggagggct tgccagttaa gagcaatgga
tactttccca 720tagaacctgg gtttgactcc cagcactaac ctacatggtg
atagtgatgc agcagacata 780catgagggca acacacacat gggcacatac
acacgcaccc gcccaccatg gcttttcccc 840catcacttag acagccatat
ttaaacgtag tggagccagg ctggggtggt ggcccacacc 900tttaatccca
gcactccaga aggcagaggt aggcggatct ctgtgggttt gagaccagcc
960tggtctacaa gagctagttc caggacagcc tccaaagcca tagagaaacc ctatc
1015111541DNACricetulus griseus 11gcctgaagac ctgagttgat acccagaacc
cagatcaaga tggaggagag aaccagcccc 60actaagctgt cccctgaccc ccataaatgc
ctccctgtcc agttatgcca cacaatgata 120ggtgaataca gaaaaacacc
cttcctttag acactaagcg gattcctctt acgcatacca 180gttaagtgat
agttcttagg cttcaactca gcactttaaa aagtttatat tttgcaatgc
240tggggactaa attagggttg tgcacatgct aagtaagcac tctacttttg
tatcacattt 300taataattgt aagaattaat tcgtgaaata gtagctgaga
caatagattt gtttctttca 360tgtgggaact gctgtgtgtg cttcttgctg
atgcaaacaa ggtcaaatac tttattcccc 420agtgtctgcc tagccctgta
acacttctct attatacaat gaccacaaat aattaggtga 480gtgggttttg
tttcatttta aattgttgct attttagaga caggatttct tgcaaacctg
540gttggtctta aactccgtat gtagctgaga atgaccttga aaaccttcct
gtcccacccc 600tcaaattcca gaattataga cacccaccac atggcttaat
aagtaaacaa caacaataaa 660agcatgactt ctgggtctgg agggagggct
tgccagttaa gagcaatgga tactttccca 720tagaacctgg gtttgactcc
cagcactaac ctacatggtg atagtgatgc agcagacata 780catgagggca
acacacacat gggcacatac acacgcaccc gcccaccatg gcttttcccc
840catcacttag acagccatat ttaaacgtag tggagccagg ctggggtggt
ggcccacacc 900tttaatccca gcactccaga aggcagaggt aggcggatct
ctgtgggttt gagaccagcc 960tggtctacaa gagctagttc caggacagcc
tccaaagcca tagagaaacc ctatctcaaa 1020aaactgaaac aacaacaaca
acaaaacaaa ataaaaaaac aacaaaagaa tcttagtggt 1080tcagtggttc
cacacacagg aaagtagaaa gggccttgat gggaaggttt tcagagggag
1140gagtatggat gagacaggat gatagtgaaa agaactcaaa ttaattaaat
atttgaaact 1200atctaagaat aaaagctaaa atatttaaaa ttacagtcag
gtagtggtgg tgcagagggc 1260taagttggta gacacagtga gatccaggcc
agccagggct acctagtgag accttgttca 1320aataactaat aaaatataca
aaataaagga gacaccacaa taattttgaa atgtaaaaga 1380ctaaatttac
cttttatatt gatgagttgg ataaaaaaat caatttacca gagaacataa
1440agtagtccca tcaaagacaa aagcaatata tgattaaact ctaatttaaa
agtttgttag 1500agcctggcaa cgtggcacat acctttaatc ccagcaccag g
1541122020DNACricetulus griseus 12gcctgaagac ctgagttgat acccagaacc
cagatcaaga tggaggagag aaccagcccc 60actaagctgt cccctgaccc ccataaatgc
ctccctgtcc agttatgcca cacaatgata 120ggtgaataca gaaaaacacc
cttcctttag acactaagcg gattcctctt acgcatacca 180gttaagtgat
agttcttagg cttcaactca gcactttaaa aagtttatat tttgcaatgc
240tggggactaa attagggttg tgcacatgct aagtaagcac tctacttttg
tatcacattt 300taataattgt aagaattaat tcgtgaaata gtagctgaga
caatagattt gtttctttca 360tgtgggaact gctgtgtgtg cttcttgctg
atgcaaacaa ggtcaaatac tttattcccc 420agtgtctgcc tagccctgta
acacttctct attatacaat gaccacaaat aattaggtga 480gtgggttttg
tttcatttta aattgttgct attttagaga caggatttct tgcaaacctg
540gttggtctta aactccgtat gtagctgaga atgaccttga aaaccttcct
gtcccacccc 600tcaaattcca gaattataga cacccaccac atggcttaat
aagtaaacaa caacaataaa 660agcatgactt ctgggtctgg agggagggct
tgccagttaa gagcaatgga tactttccca 720tagaacctgg gtttgactcc
cagcactaac ctacatggtg atagtgatgc agcagacata 780catgagggca
acacacacat gggcacatac acacgcaccc gcccaccatg gcttttcccc
840catcacttag acagccatat ttaaacgtag tggagccagg ctggggtggt
ggcccacacc 900tttaatccca gcactccaga aggcagaggt aggcggatct
ctgtgggttt gagaccagcc 960tggtctacaa gagctagttc caggacagcc
tccaaagcca tagagaaacc ctatctcaaa 1020aaactgaaac aacaacaaca
acaaaacaaa ataaaaaaac aacaaaagaa tcttagtggt 1080tcagtggttc
cacacacagg aaagtagaaa gggccttgat gggaaggttt tcagagggag
1140gagtatggat gagacaggat gatagtgaaa agaactcaaa ttaattaaat
atttgaaact 1200atctaagaat aaaagctaaa atatttaaaa ttacagtcag
gtagtggtgg tgcagagggc 1260taagttggta gacacagtga gatccaggcc
agccagggct acctagtgag accttgttca 1320aataactaat aaaatataca
aaataaagga gacaccacaa taattttgaa atgtaaaaga 1380ctaaatttac
cttttatatt gatgagttgg ataaaaaaat caatttacca gagaacataa
1440agtagtccca tcaaagacaa aagcaatata tgattaaact ctaatttaaa
agtttgttag 1500agcctggcaa cgtggcacat acctttaatc ccagcaccag
ggagacagag gccatcctgg 1560tctaaaaagt gatctccagg acagccatgg
ctattacaca gagaaaccct gtctggaaaa 1620acaaaaaatt agtgtccatg
tgtaaatgtg tggagtatgc ttgtcatgcc acatacagag 1680gtagagggca
gtttatggga gtcagttcct attcttcctt tatgggggac ctggggactg
1740aactcaggtc atcaggcttg gcagaaagtg cattagctca cggagcctta
tcattggcga 1800aagctctctc aagtagaaaa tcaatgtgtt tgctcatagt
gcaatcatta tgtttcgaga 1860ggggaagggt acaatcgttg gggcatgtgt
ggtcacatct gaatagcagt agctccctag 1920gagaattaat tccaagttct
ttggtggtgt atcaatgccc ttaaaggggt caacaacttt 1980ttttccctct
gacaaaacta tcttcttatg tccttgtccc 2020132516DNACricetulus griseus
13gcctgaagac ctgagttgat acccagaacc cagatcaaga tggaggagag aaccagcccc
60actaagctgt cccctgaccc ccataaatgc ctccctgtcc agttatgcca cacaatgata
120ggtgaataca gaaaaacacc cttcctttag acactaagcg gattcctctt
acgcatacca 180gttaagtgat agttcttagg cttcaactca gcactttaaa
aagtttatat tttgcaatgc 240tggggactaa attagggttg tgcacatgct
aagtaagcac tctacttttg tatcacattt 300taataattgt aagaattaat
tcgtgaaata gtagctgaga caatagattt gtttctttca 360tgtgggaact
gctgtgtgtg cttcttgctg atgcaaacaa ggtcaaatac tttattcccc
420agtgtctgcc tagccctgta acacttctct attatacaat gaccacaaat
aattaggtga 480gtgggttttg tttcatttta aattgttgct attttagaga
caggatttct tgcaaacctg 540gttggtctta aactccgtat gtagctgaga
atgaccttga aaaccttcct gtcccacccc 600tcaaattcca gaattataga
cacccaccac atggcttaat aagtaaacaa caacaataaa 660agcatgactt
ctgggtctgg agggagggct tgccagttaa gagcaatgga tactttccca
720tagaacctgg gtttgactcc cagcactaac ctacatggtg atagtgatgc
agcagacata 780catgagggca acacacacat gggcacatac acacgcaccc
gcccaccatg gcttttcccc 840catcacttag acagccatat ttaaacgtag
tggagccagg ctggggtggt ggcccacacc 900tttaatccca gcactccaga
aggcagaggt aggcggatct ctgtgggttt gagaccagcc 960tggtctacaa
gagctagttc caggacagcc tccaaagcca tagagaaacc ctatctcaaa
1020aaactgaaac aacaacaaca acaaaacaaa ataaaaaaac aacaaaagaa
tcttagtggt 1080tcagtggttc cacacacagg aaagtagaaa gggccttgat
gggaaggttt tcagagggag 1140gagtatggat gagacaggat gatagtgaaa
agaactcaaa ttaattaaat atttgaaact 1200atctaagaat aaaagctaaa
atatttaaaa ttacagtcag gtagtggtgg tgcagagggc 1260taagttggta
gacacagtga gatccaggcc agccagggct acctagtgag accttgttca
1320aataactaat aaaatataca aaataaagga gacaccacaa taattttgaa
atgtaaaaga 1380ctaaatttac cttttatatt gatgagttgg ataaaaaaat
caatttacca gagaacataa 1440agtagtccca tcaaagacaa aagcaatata
tgattaaact ctaatttaaa agtttgttag 1500agcctggcaa cgtggcacat
acctttaatc ccagcaccag ggagacagag gccatcctgg 1560tctaaaaagt
gatctccagg acagccatgg ctattacaca gagaaaccct gtctggaaaa
1620acaaaaaatt agtgtccatg tgtaaatgtg tggagtatgc ttgtcatgcc
acatacagag 1680gtagagggca gtttatggga gtcagttcct attcttcctt
tatgggggac ctggggactg 1740aactcaggtc atcaggcttg gcagaaagtg
cattagctca cggagcctta tcattggcga 1800aagctctctc aagtagaaaa
tcaatgtgtt tgctcatagt gcaatcatta tgtttcgaga 1860ggggaagggt
acaatcgttg gggcatgtgt ggtcacatct gaatagcagt agctccctag
1920gagaattaat tccaagttct ttggtggtgt atcaatgccc ttaaaggggt
caacaacttt 1980ttttccctct gacaaaacta tcttcttatg tccttgtccc
tcatatttga agtattttat 2040tctttgcagt gttgaatatc aattctagca
cctcagacat gttaggtaag taccctacaa 2100ctcaggttaa ctaatttaat
ttaactaatt taaccccaac actttttctt tgtttatcca 2160catttgtgga
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
2220gtgtgtgtgt gtgcgcgcgc gcgcgcgctc ggatcattct accttttgtt
taaaaaatgt 2280tagtccaggg gtggggtgca ctgtgaaagt ctgagggtaa
cttgctgggg tcagttcttt 2340ccactatagg acagaactcc aggtgtcaac
tctttactga cagaaccatc caaatagccc 2400tatctaattt tagtttttta
tttatttatt ttttgttttt cgagacaggg tttctctgtg 2460gctttggagg
ctgtcctgga actagctctt gtagaccagg ctggtctcga actcag
2516143148DNACricetulus griseus 14gcctgaagac ctgagttgat acccagaacc
cagatcaaga tggaggagag aaccagcccc 60actaagctgt cccctgaccc ccataaatgc
ctccctgtcc agttatgcca cacaatgata 120ggtgaataca gaaaaacacc
cttcctttag acactaagcg gattcctctt acgcatacca 180gttaagtgat
agttcttagg cttcaactca gcactttaaa aagtttatat tttgcaatgc
240tggggactaa attagggttg tgcacatgct aagtaagcac tctacttttg
tatcacattt 300taataattgt aagaattaat tcgtgaaata gtagctgaga
caatagattt gtttctttca 360tgtgggaact gctgtgtgtg cttcttgctg
atgcaaacaa ggtcaaatac tttattcccc 420agtgtctgcc tagccctgta
acacttctct attatacaat gaccacaaat aattaggtga 480gtgggttttg
tttcatttta aattgttgct attttagaga caggatttct tgcaaacctg
540gttggtctta aactccgtat gtagctgaga atgaccttga aaaccttcct
gtcccacccc 600tcaaattcca gaattataga cacccaccac atggcttaat
aagtaaacaa caacaataaa 660agcatgactt ctgggtctgg agggagggct
tgccagttaa gagcaatgga tactttccca 720tagaacctgg gtttgactcc
cagcactaac ctacatggtg atagtgatgc agcagacata 780catgagggca
acacacacat gggcacatac acacgcaccc gcccaccatg gcttttcccc
840catcacttag acagccatat ttaaacgtag tggagccagg ctggggtggt
ggcccacacc 900tttaatccca gcactccaga aggcagaggt aggcggatct
ctgtgggttt gagaccagcc 960tggtctacaa gagctagttc caggacagcc
tccaaagcca tagagaaacc ctatctcaaa 1020aaactgaaac aacaacaaca
acaaaacaaa ataaaaaaac aacaaaagaa tcttagtggt 1080tcagtggttc
cacacacagg aaagtagaaa gggccttgat gggaaggttt tcagagggag
1140gagtatggat gagacaggat gatagtgaaa agaactcaaa ttaattaaat
atttgaaact 1200atctaagaat aaaagctaaa atatttaaaa ttacagtcag
gtagtggtgg tgcagagggc 1260taagttggta gacacagtga gatccaggcc
agccagggct acctagtgag accttgttca 1320aataactaat aaaatataca
aaataaagga gacaccacaa taattttgaa atgtaaaaga 1380ctaaatttac
cttttatatt gatgagttgg ataaaaaaat caatttacca gagaacataa
1440agtagtccca tcaaagacaa aagcaatata tgattaaact ctaatttaaa
agtttgttag 1500agcctggcaa cgtggcacat acctttaatc ccagcaccag
ggagacagag gccatcctgg 1560tctaaaaagt gatctccagg acagccatgg
ctattacaca gagaaaccct gtctggaaaa 1620acaaaaaatt agtgtccatg
tgtaaatgtg tggagtatgc ttgtcatgcc acatacagag 1680gtagagggca
gtttatggga gtcagttcct attcttcctt tatgggggac ctggggactg
1740aactcaggtc atcaggcttg gcagaaagtg cattagctca cggagcctta
tcattggcga 1800aagctctctc aagtagaaaa tcaatgtgtt tgctcatagt
gcaatcatta tgtttcgaga 1860ggggaagggt acaatcgttg gggcatgtgt
ggtcacatct gaatagcagt agctccctag 1920gagaattaat tccaagttct
ttggtggtgt atcaatgccc ttaaaggggt caacaacttt 1980ttttccctct
gacaaaacta tcttcttatg tccttgtccc tcatatttga agtattttat
2040tctttgcagt gttgaatatc aattctagca cctcagacat gttaggtaag
taccctacaa 2100ctcaggttaa ctaatttaat ttaactaatt taaccccaac
actttttctt tgtttatcca 2160catttgtgga gtgtgtgtgt gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 2220gtgtgtgtgt gtgcgcgcgc
gcgcgcgctc ggatcattct accttttgtt taaaaaatgt 2280tagtccaggg
gtggggtgca ctgtgaaagt ctgagggtaa cttgctgggg tcagttcttt
2340ccactatagg acagaactcc aggtgtcaac tctttactga cagaaccatc
caaatagccc 2400tatctaattt tagtttttta tttatttatt ttttgttttt
cgagacaggg tttctctgtg 2460gctttggagg ctgtcctgga actagctctt
gtagaccagg ctggtctcga actcagagat 2520ccacctgcct ctgcctcctg
agtgctggga ttaaaggcat gcgccaccaa cgcttggctc 2580tacctaattt
taaaagagat tgtgtgtcac aagggtgtca tgtcgccctg caaccacccc
2640ccccccaaaa aaaaaaaaaa aaaaacttca ctgaagctga agcacgatga
tttggttact 2700ctggctggcc aatgagctct agggagtctc ctgtcaaaca
gaatctcaac aggcgcagca 2760gtctttttta aagtggggtt acaacacagg
tttttgcata tcaggcattt tatctaagct 2820atttcccagc caaaaatgtg
tattttggag gcagcagagc taatagatta aaatgaggga 2880agagcccaca
caggttatta ggaagataag catcttcttt atataaaaca aaaccaaacc
2940aaactggagg aggtctacct ttagggatgg aagaaaagac atttagaggg
tgcaatagaa 3000agggcactga gtttgtgagg tggaggactg ggagagggcg
caaccgcttt aactgtcctg 3060ttttgcctat tttttgggga cagcacatgt
tcctattttt cccaggatgg gcaatctcca 3120cgtccaaact tgcggtcgag gactacag
314815511DNACricetulus griseus 15gttgctattt tagagacagg atttcttgca
aacctggttg gtcttaaact ccgtatgtag 60ctgagaatga ccttgaaaac cttcctgtcc
cacccctcaa attccagaat tatagacacc 120caccacatgg cttaataagt
aaacaacaac aataaaagca tgacttctgg gtctggaggg 180agggcttgcc
agttaagagc aatggatact ttcccataga acctgggttt gactcccagc
240actaacctac atggtgatag tgatgcagca gacatacatg agggcaacac
acacatgggc 300acatacacac gcacccgccc accatggctt ttcccccatc
acttagacag ccatatttaa 360acgtagtgga gccaggctgg ggtggtggcc
cacaccttta atcccagcac tccagaaggc 420agaggtaggc ggatctctgt
gggtttgaga ccagcctggt ctacaagagc tagttccagg 480acagcctcca
aagccataga gaaaccctat c 51116549DNACricetulus griseus 16caaagccata
gagaaaccct atctcaaaaa actgaaacaa caacaacaac aaaacaaaat 60aaaaaaacaa
caaaagaatc ttagtggttc agtggttcca cacacaggaa agtagaaagg
120gccttgatgg gaaggttttc agagggagga gtatggatga gacaggatga
tagtgaaaag 180aactcaaatt aattaaatat ttgaaactat ctaagaataa
aagctaaaat atttaaaatt 240acagtcaggt agtggtggtg cagagggcta
agttggtaga cacagtgaga tccaggccag 300ccagggctac ctagtgagac
cttgttcaaa taactaataa aatatacaaa ataaaggaga 360caccacaata
attttgaaat gtaaaagact aaatttacct tttatattga tgagttggat
420aaaaaaatca atttaccaga gaacataaag tagtcccatc aaagacaaaa
gcaatatatg 480attaaactct aatttaaaag tttgttagag cctggcaacg
tggcacatac ctttaatccc 540agcaccagg 549171037DNACricetulus griseus
17gttgctattt tagagacagg atttcttgca aacctggttg gtcttaaact ccgtatgtag
60ctgagaatga ccttgaaaac cttcctgtcc cacccctcaa attccagaat tatagacacc
120caccacatgg cttaataagt aaacaacaac aataaaagca tgacttctgg
gtctggaggg 180agggcttgcc agttaagagc aatggatact ttcccataga
acctgggttt gactcccagc 240actaacctac atggtgatag tgatgcagca
gacatacatg agggcaacac acacatgggc 300acatacacac gcacccgccc
accatggctt ttcccccatc acttagacag ccatatttaa 360acgtagtgga
gccaggctgg ggtggtggcc cacaccttta atcccagcac tccagaaggc
420agaggtaggc ggatctctgt gggtttgaga ccagcctggt ctacaagagc
tagttccagg 480acagcctcca aagccataga gaaaccctat ctcaaaaaac
tgaaacaaca acaacaacaa 540aacaaaataa aaaaacaaca aaagaatctt
agtggttcag tggttccaca cacaggaaag 600tagaaagggc cttgatggga
aggttttcag agggaggagt atggatgaga caggatgata 660gtgaaaagaa
ctcaaattaa ttaaatattt gaaactatct aagaataaaa gctaaaatat
720ttaaaattac agtcaggtag tggtggtgca
gagggctaag ttggtagaca cagtgagatc 780caggccagcc agggctacct
agtgagacct tgttcaaata actaataaaa tatacaaaat 840aaaggagaca
ccacaataat tttgaaatgt aaaagactaa atttaccttt tatattgatg
900agttggataa aaaaatcaat ttaccagaga acataaagta gtcccatcaa
agacaaaagc 960aatatatgat taaactctaa tttaaaagtt tgttagagcc
tggcaacgtg gcacatacct 1020ttaatcccag caccagg 103718500DNACricetulus
griseus 18acctttaatc ccagcaccag ggagacagag gccatcctgg tctaaaaagt
gatctccagg 60acagccatgg ctattacaca gagaaaccct gtctggaaaa acaaaaaatt
agtgtccatg 120tgtaaatgtg tggagtatgc ttgtcatgcc acatacagag
gtagagggca gtttatggga 180gtcagttcct attcttcctt tatgggggac
ctggggactg aactcaggtc atcaggcttg 240gcagaaagtg cattagctca
cggagcctta tcattggcga aagctctctc aagtagaaaa 300tcaatgtgtt
tgctcatagt gcaatcatta tgtttcgaga ggggaagggt acaatcgttg
360gggcatgtgt ggtcacatct gaatagcagt agctccctag gagaattaat
tccaagttct 420ttggtggtgt atcaatgccc ttaaaggggt caacaacttt
ttttccctct gacaaaacta 480tcttcttatg tccttgtccc
500191028DNACricetulus griseus 19caaagccata gagaaaccct atctcaaaaa
actgaaacaa caacaacaac aaaacaaaat 60aaaaaaacaa caaaagaatc ttagtggttc
agtggttcca cacacaggaa agtagaaagg 120gccttgatgg gaaggttttc
agagggagga gtatggatga gacaggatga tagtgaaaag 180aactcaaatt
aattaaatat ttgaaactat ctaagaataa aagctaaaat atttaaaatt
240acagtcaggt agtggtggtg cagagggcta agttggtaga cacagtgaga
tccaggccag 300ccagggctac ctagtgagac cttgttcaaa taactaataa
aatatacaaa ataaaggaga 360caccacaata attttgaaat gtaaaagact
aaatttacct tttatattga tgagttggat 420aaaaaaatca atttaccaga
gaacataaag tagtcccatc aaagacaaaa gcaatatatg 480attaaactct
aatttaaaag tttgttagag cctggcaacg tggcacatac ctttaatccc
540agcaccaggg agacagaggc catcctggtc taaaaagtga tctccaggac
agccatggct 600attacacaga gaaaccctgt ctggaaaaac aaaaaattag
tgtccatgtg taaatgtgtg 660gagtatgctt gtcatgccac atacagaggt
agagggcagt ttatgggagt cagttcctat 720tcttccttta tgggggacct
ggggactgaa ctcaggtcat caggcttggc agaaagtgca 780ttagctcacg
gagccttatc attggcgaaa gctctctcaa gtagaaaatc aatgtgtttg
840ctcatagtgc aatcattatg tttcgagagg ggaagggtac aatcgttggg
gcatgtgtgg 900tcacatctga atagcagtag ctccctagga gaattaattc
caagttcttt ggtggtgtat 960caatgccctt aaaggggtca acaacttttt
ttccctctga caaaactatc ttcttatgtc 1020cttgtccc
1028201516DNACricetulus griseus 20gttgctattt tagagacagg atttcttgca
aacctggttg gtcttaaact ccgtatgtag 60ctgagaatga ccttgaaaac cttcctgtcc
cacccctcaa attccagaat tatagacacc 120caccacatgg cttaataagt
aaacaacaac aataaaagca tgacttctgg gtctggaggg 180agggcttgcc
agttaagagc aatggatact ttcccataga acctgggttt gactcccagc
240actaacctac atggtgatag tgatgcagca gacatacatg agggcaacac
acacatgggc 300acatacacac gcacccgccc accatggctt ttcccccatc
acttagacag ccatatttaa 360acgtagtgga gccaggctgg ggtggtggcc
cacaccttta atcccagcac tccagaaggc 420agaggtaggc ggatctctgt
gggtttgaga ccagcctggt ctacaagagc tagttccagg 480acagcctcca
aagccataga gaaaccctat ctcaaaaaac tgaaacaaca acaacaacaa
540aacaaaataa aaaaacaaca aaagaatctt agtggttcag tggttccaca
cacaggaaag 600tagaaagggc cttgatggga aggttttcag agggaggagt
atggatgaga caggatgata 660gtgaaaagaa ctcaaattaa ttaaatattt
gaaactatct aagaataaaa gctaaaatat 720ttaaaattac agtcaggtag
tggtggtgca gagggctaag ttggtagaca cagtgagatc 780caggccagcc
agggctacct agtgagacct tgttcaaata actaataaaa tatacaaaat
840aaaggagaca ccacaataat tttgaaatgt aaaagactaa atttaccttt
tatattgatg 900agttggataa aaaaatcaat ttaccagaga acataaagta
gtcccatcaa agacaaaagc 960aatatatgat taaactctaa tttaaaagtt
tgttagagcc tggcaacgtg gcacatacct 1020ttaatcccag caccagggag
acagaggcca tcctggtcta aaaagtgatc tccaggacag 1080ccatggctat
tacacagaga aaccctgtct ggaaaaacaa aaaattagtg tccatgtgta
1140aatgtgtgga gtatgcttgt catgccacat acagaggtag agggcagttt
atgggagtca 1200gttcctattc ttcctttatg ggggacctgg ggactgaact
caggtcatca ggcttggcag 1260aaagtgcatt agctcacgga gccttatcat
tggcgaaagc tctctcaagt agaaaatcaa 1320tgtgtttgct catagtgcaa
tcattatgtt tcgagagggg aagggtacaa tcgttggggc 1380atgtgtggtc
acatctgaat agcagtagct ccctaggaga attaattcca agttctttgg
1440tggtgtatca atgcccttaa aggggtcaac aacttttttt ccctctgaca
aaactatctt 1500cttatgtcct tgtccc 151621381DNACricetulus griseus
21cttgcggtcg aggactacag tcattttgca ggtttcctta ctgtatggct tttaaaacgt
60gcaaaggtga ccattaaccg tttcacgctg ggagggcacg tgcggctcag atgcttcctc
120tgactgaggg ccaggagggg gctacacgga agaggccaca cccgcacttg
ggaagactcg 180atttgggctt cagctggctg agacgcccca gcaggctcct
cggctacacc ttcagccccg 240aatgccttcc ggcccataac ccttcccttc
taggcatttc cggcgaggac ccaccctcgc 300gccaaacatt cggccccatc
ccccggtcct cacctgaatc tctaactctg actccagagt 360ttagagacta
taaccagata g 3812234DNAArtificialprimer 22ctatgaggat ccgcctgaag
acctgagttg atac 342337DNAArtificialprimer 23tatgcaggat ccgttgctat
tttagagaca ggatttc 372435DNAArtificialprimer 24tatgcaggat
cccaaagcca tagagaaacc ctatc 352533DNAArtificialprimer 25tatgcaggat
ccacctttaa tcccagcacc agg 332635DNAArtificialprimer 26ctatgaggat
ccctatcttc ttatgtcctt gtccc 352732DNAArtificialprimer 27tatgcaggat
cccaggctgg tctcgaactc ag 322832DNAArtificialprimer 28ctatgaggat
cccttgcggt cgaggactac ag 322934DNAArtificialprimer 29ctatgatgta
cagcctgaag acctgagttg atac 343039DNAArtificialprimer 30attgcatgta
cactatctgg ttatagtctc taaactctg 393137DNAArtificialprimer
31atagcatgta cagaaatcct gtctctaaaa tagcaac
373235DNAArtificialprimer 32atagcatgta cagatagggt ttctctatgg ctttg
353333DNAArtificialprimer 33atacgatgta cacctggtgc tgggattaaa ggt
333435DNAArtificialprimer 34atagcatgta cagggacaag gacataagaa gatag
353532DNAArtificialprimer 35tagttatgta cactgagttc gagaccagcc tg
323632DNAArtificialprimer 36atagcatgta cactgtagtc ctcgaccgca ag
323733DNAArtificialprimer 37atacgaggat cccctggtgc tgggattaaa ggt
333835DNAArtificialprimer 38atagcaggat ccgatagggt ttctctatgg ctttg
353924DNAArtificialprimer 39ctccacacat ttacacatgg acac
244024DNAArtificialprimer 40gggtttctct gtgtaatagc catg
244124DNAArtificialprimer 41atctcactgt gtctaccaac ttag
244222DNAArtificialprimer 42tctgcaccac cactacctga ct
224328DNAArtificialprimer 43ctaagagtac ttgccatgag agcctgaa
284425DNAArtificialprimer 44cattgataca ccaccaaaga acttg 25
* * * * *